Stabilized liquid polypeptide-containing pharmaceutical compositions

ABSTRACT

Stabilized liquid polypeptide-containing pharmaceutical compositions are provided. The compositions comprise an amino acid base, which serves as the primary stabilizing agent of the polypeptide, and an acid and/or its salt form to buffer the solution within an acceptable pH range for stability of the polypeptide. Methods for increasing stability of a polypeptide in a liquid pharmaceutical composition and for increasing storage stability of such a pharmaceutical composition are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/299,039, filed Nov. 18, 2002, now allowed, which is a continuation ofU.S. application Ser. No. 09/677,643, filed Oct. 3, 2000, now U.S. Pat.No. 6,525,102, which claims the benefit of priority of U.S. ProvisionalApplication Ser. No. 60/157,696, filed Oct. 4, 1999, each of which ishereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to pharmaceutical compositions,more particularly to pharmaceutical compositions comprising polypeptidesthat typically are unstable in liquid pharmaceutical formulations.

BACKGROUND OF THE INVENTION

Recent advances in the development of genetic engineering technologyhave provided a wide variety of biologically active polypeptides insufficiently large quantities for use as drugs. Polypeptides, however,can lose biological activity as a result of physical instabilities,including denaturation and formation of soluble and insolubleaggregates, and a variety of chemical instabilities, such as hydrolysis,oxidation, and deamidation. Stability of polypeptides in liquidpharmaceutical formulations can be affected, for example, by factorssuch as pH, ionic strength, temperature, repeated cycles of freeze-thaw,and exposure to mechanical shear forces such as occur during processing.Aggregate formation and loss of biological activity can also occur as aresult of physical agitation and interactions of polypeptide moleculesin solution and at the liquid-air interfaces within storage vials.Further conformational changes may occur in polypeptides adsorbed toair-liquid and solid-liquid interfaces during compression-extension ofthe interfaces resulting from agitation during transportation orotherwise. Such agitation can cause the protein to entangle, aggregate,form particles, and ultimately precipitate with other adsorbed proteins.For a general review of stability of protein pharmaceuticals, see, forexample, Manning et al. (1989) Pharm. Res. 6:903-918, and Wang andHanson (1988) J. Parenteral Sci. Tech. 42:S14.

Instability of polypeptide-containing liquid pharmaceutical formulationshas prompted packaging of these formulations in the lyophilized formalong with a suitable liquid medium for reconstitution. Althoughlyophilization improves storage stability of the composition, manypolypeptides exhibit decreased activity, either during storage in thedried state (Pikal (1990) Biopharm. 27:26-30) or as a result ofaggregate formation or loss of catalytic activity upon reconstitution asa liquid formulation (see, for example, Carpenter et al. (1991) Develop.Biol. Standard 74:225-239; Broadhead et al. (1992) Drug Devel. Ind.Pharm. 18:1169-1206; Mumenthaler et al. (1994) Pharm. Res. 11:12-20;Carpenter and Crowe (1988) Cryobiology 25:459-470; and Roser (1991)Biopharm. 4:47-53). While the use of additives has improved thestability of dried proteins, many rehydrated formulations continue tohave unacceptable or undesirable amounts of inactive, aggregated protein(see, for example, Townsend and DeLuca (1983) J. Pharm. Sci. 80:63-66;Hora et al. (1992) Pharm. Res. 9:33-36; Yoshiaka et al. (1993) Pharm.Res, 10:687-691). Also, the need for reconstitution is an inconvenienceand introduces the possibility of incorrect dosing.

While a number of liquid pharmaceutical compositions have beenformulated to stabilize the biological activity of polypeptidescontained therein, the degradation of polypeptides in liquidformulations continues to create problems for medical practitioners.Consequently, there is a need for additional pharmaceutical compositionscomprising physiologically compatible stabilizers that promote stabilityof polypeptide components, thereby maintaining their therapeuticeffectiveness.

SUMMARY OF THE INVENTION

Compositions comprising a polypeptide as a therapeutically activecomponent and methods useful in their preparation are provided. Thecompositions are stabilized liquid pharmaceutical compositions thatinclude a polypeptide whose effectiveness as a therapeutically activecomponent is normally compromised during storage in liquid formulationsas a result of aggregation of the polypeptide. The stabilized liquidpharmaceutical compositions of the invention comprise, in addition to apolypeptide that exhibits aggregate formation during storage in a liquidformulation, an amount of an amino acid base sufficient to decreaseaggregate formation of the polypeptide during storage, where the aminoacid base is an amino acid or a combination of amino acids, where anygiven amino acid is present either in its free base form or in its saltform. The compositions further comprise a buffering agent to maintain pHof the liquid composition within an acceptable range for stability ofthe polypeptide, where the buffering agent is an acid substantially freeof its salt form, an acid in its salt form, or a mixture of an acid andits salt form.

The amino acid base serves to stabilize the polypeptide againstaggregate formation during storage of the liquid pharmaceuticalcomposition, while use of an acid substantially free of its salt form,an acid in its salt form, or a mixture of an acid and its salt form asthe buffering agent results in a liquid composition having an osmolaritythat is nearly isotonic. The liquid pharmaceutical composition mayadditionally incorporate other stabilizing agents, more particularlymethionine, a nonionic surfactant such as polysorbate 80, and EDTA, tofurther increase stability of the polypeptide. Such liquidpharmaceutical compositions are said to be stabilized, as addition ofamino acid base in combination with an acid substantially free of itssalt form, an acid in its salt form, or a mixture of an acid and itssalt form, results in the compositions having increased storagestability relative to liquid pharmaceutical compositions formulated inthe absence of the combination of these two components.

Methods for increasing stability of a polypeptide in a liquidpharmaceutical composition and for increasing storage stability of sucha pharmaceutical composition are also provided. The methods compriseincorporating into the liquid pharmaceutical composition an amount of anamino acid base sufficient to decrease aggregate formation of thepolypeptide during storage of the composition, and a buffering agent,where the buffering agent is an acid substantially free of its saltform, an acid in its salt form, or a mixture of an acid and its saltform. The methods find use in preparation of the liquid pharmaceuticalcompositions of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the percent remaining of soluble IL-2 in stability samplesstored at 40° C., as analyzed by RP-HPLC. Formulations contained 0.2mg/ml IL-2, 10 mM sodium succinate at pH 6, and 270 mM sorbitol orsucrose or mannitol.

FIG. 2 shows the percent remaining of soluble IL-2 in stability samplesstored at 50° C., as analyzed by RP-HPLC. Formulations contained 0.1mg/ml IL-2, 10 mM sodium succinate at pH 6, and 150 mM of various aminoacids as indicated in the figure.

FIG. 3 shows the percent remaining of soluble IL-2 in stability samplesstored at 40° C., as analyzed by RP-HPLC. Formulations contained 0.2mg/ml IL-2, 10 mM sodium succinate at pH 6, and 50, 100, or 270 mMsorbitol.

FIG. 4 shows the percent remaining of soluble IL-2 in stability samplesstored at 50° C., as analyzed by RP-HPLC. Formulations contained 0.2mg/ml IL-2, 10 mM sodium succinate at pH 6, and 50, 100, or 150 mMarginine.

FIG. 5 shows the half-life (t_(1/2), in days) of remaining soluble IL-2analyzed by RP-HPLC as a function of pH at 50° C. Formulations contained0.2 mg/ml IL-2, 10 mM buffer (glycine, sodium acetate, sodium citrate,sodium succinate, sodium phosphate, sodium borate), and 150 mM NaCl, 270mM sorbitol, or 150 mM arginine.

FIG. 6 shows the Ln-Ln plot of half-life (t_(1/2)) versus initialprotein concentration for stability samples stored at 50° C.Formulations contained 0.1, 0.2, or 0.5 mg/ml IL-2 in 10 mM sodiumsuccinate at pH 6 and 150 mM L-arginine.

FIG. 7 shows the percent remaining of soluble IL-2, as analyzed byRP-HPLC, in samples treated with 1, 3, and 5 cycles of freeze-thaw from−70° C. to ambient temperature. Formulations contained 0.2 mg/ml IL-2,10 mM sodium succinate at pH 6, 150 mM arginine, and 0 to 0.1%polysorbate 80.

FIG. 8 shows the percent remaining of soluble IL-2, as analyzed byRP-HPLC, in samples treated with shipment from Emeryville, Calif., toSt. Louis, Mo., and from St. Louis back to Emeryville on ice. Twoformulations containing various amount of polysorbate 80 were used: anarginine formulation, containing 0.2 mg/ml IL-2 in 10 mM sodiumsuccinate at pH 6 and 150 mM arginine; and a NaCl formulation,containing 0.2 mg/ml IL-2 in 10 mM sodium citrate at pH 6.5 and 200 mMNaCl.

FIG. 9 shows the half-life (t_(1/2), in days) of remaining soluble TFPIin four formulations analyzed by IEX-HPLC as a function of arginineconcentration at 50° C. All formulations contained 0.15 mg/ml TFPI andeither L-arginine base or L-arginine HCl, buffered to pH 5.5 with eithercitric acid or 10 mM citric acid and sodium citrate. The specific TFPIformulations contained: (a) 20-150 mM L-arginine HCl, 10 mM citric acidand sodium citrate as buffer; (b) 20-150 mM L-arginine base, titratedwith citric acid; (c) 100-300 mM L-arginine HCl, 10 mM citric acid andsodium citrate as buffer; (d) 100-300 mM L-arginine base titrated withcitric acid.

FIG. 10 shows the half-life (t_(1/2), in days) of remaining soluble TFPIin four formulations analyzed by IEX-HPLC as a function of arginineconcentration at 50° C. All formulations contained 0.15 mg/ml TFPI andeither L-arginine base or L-arginine HCl, buffered to pH 5.5 with eithersuccinic acid or 10 mM succinic acid and sodium succinate. The specificTFPI formulations contained: (a) 20-150 mM L-arginine HCl, 10 mMsuccinic acid and sodium succinate as buffer; (b) 20-150 mM L-argininebase, titrated with succinic acid; (c) 100-300 mM L-arginine HCl, 10 mMsuccinic acid and sodium succinate as buffer; and (d) 100-300 mML-arginine base titrated with succinic acid.

FIG. 11 shows the half-life (t_(1/2), in days) of remaining soluble TFPIin four formulations analyzed by IEX-HPLC as a function of arginineconcentration at 50° C. All formulations contained 0.15 mg/ml TFPI andL-arginine base, titrated to pH 5.5 with either succinic acid or citricacid. The specific TFPI formulations contained: (a) 20-150 mM L-argininebase, titrated with citric acid; (b) 20-150 mM L-arginine base, titratedwith succinic acid; (c) 100-300 mM L-arginine base titrated with citricacid; (d) 100-300 mM L-arginine base titrated with succinic acid.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to liquid pharmaceutical compositionscomprising a polypeptide as a therapeutically active component and tomethods useful in their preparation. For purposes of the presentinvention, the term “liquid” with regard to pharmaceutical compositionsor formulations is intended to include the term “aqueous”. The term“polypeptide” as used herein encompasses naturally occurring (native),synthetic, and recombinant polypeptides and proteins, and biologicallyactive variants thereof, as qualified elsewhere herein. By“therapeutically active component” is intended the polypeptide isspecifically incorporated into the composition to bring about a desiredtherapeutic response with regard to treatment, prevention, or diagnosisof a disease or condition within a subject when the pharmaceuticalcomposition is administered to that subject.

More particularly, compositions of the invention are stabilized liquidpharmaceutical compositions whose therapeutically active componentsinclude a polypeptide that normally exhibits aggregate formation duringstorage in liquid pharmaceutical formulations. By “aggregate formation”is intended a physical interaction between the polypeptide moleculesthat results in formation of oligomers, which may remain soluble, orlarge visible aggregates that precipitate out of solution. By “duringstorage” is intended a liquid pharmaceutical composition or formulationonce prepared, is not immediately administered to a subject. Rather,following preparation, it is packaged for storage, either in a liquidform, in a frozen state, or in a dried form for later reconstitutioninto a liquid form or other form suitable for administration to asubject. By “dried form” is intended the liquid pharmaceuticalcomposition or formulation is dried either by freeze drying (i.e.,lyophilization; see, for example, Williams and Polli (1984) J.Parenteral Sci. Technol. 38:48-59), spray drying (see Masters (1991) inSpray-Drying Handbook (5th ed; Longman Scientific and Technical, Essez,U.K.), pp. 491-676; Broadhead et al. (1992) Drug Devel. Ind. Pharm.18:1169-1206; and Mumenthaler et al. (1994) Pharm. Res. 11:12-20), orair drying (Carpenter and Crowe (1988) Cryobiology 25:459-470; and Roser(1991) Biopharm. 4:47-53). Aggregate formation by a polypeptide duringstorage of a liquid pharmaceutical composition can adversely affectbiological activity of that polypeptide, resulting in loss oftherapeutic efficacy of the pharmaceutical composition. Furthermore,aggregate formation may cause other problems such as blockage of tubing,membranes, or pumps when the polypeptide-containing pharmaceuticalcomposition is administered using an infusion system.

The stabilized liquid pharmaceutical compositions of the inventionfurther comprise an amount of an amino acid base sufficient to decreaseaggregate formation by the polypeptide during storage of thecomposition. By “amino acid base” is intended an amino acid or acombination of amino acids, where any given amino acid is present eitherin its free base form or in its salt form. Where a combination of aminoacids is used, all of the amino acids may be present in their free baseforms, all may be present in their salt forms, or some may be present intheir free base forms while others are present in their salt forms.Preferred amino acids to use in preparing the compositions of theinvention are those carrying a charged side chain, such as arginine,lysine, aspartic acid, and glutamic acid. Any stereoisomer (i.e., L, D,or DL isomer) of a particular amino acid, or combinations of thesestereoisomers, may be present in the pharmaceutical compositions of theinvention so long as the particular amino acid is present either in itsfree base form or its salt form. Preferably the L-stereoisomer is used.Compositions of the invention may also be formulated with analogues ofthese preferred amino acids. By “amino acid analogue” is intended aderivative of the naturally occurring amino acid that brings about thedesired effect of decreasing aggregate formation by the polypeptideduring storage of the liquid pharmaceutical compositions of theinvention. Suitable arginine analogues include, for example,aminoguanidine and N-monoethyl L-arginine. As with the preferred aminoacids, the amino acid analogues are incorporated into the compositionsin either their free base form or their salt form.

In combination with the amino acid base as defined herein, thestabilized liquid pharmaceutical compositions of the invention furthercomprise an acid substantially free of its salt form, an acid in itssalt form, or a mixture of an acid and its salt form to maintainsolution pH. Preferably, the pH is maintained by using the amino acidbase in combination with an acid substantially free of its salt form.Such a combination provides for a lower osmolarity of the solution thanif an acid and its salt form are used as buffering agents in combinationwith an amino acid base to formulate a stabilized pharmaceuticalcomposition. The advantage of such a combination is that one canincorporate a higher concentration of the stabilizer, the amino acidbase, into the pharmaceutical composition without exceeding isotonicityof the solution. By “an acid substantially free of its salt form” isintended that the acid serving as the buffering agent within the liquidpharmaceutical composition is present in the absence of any of its saltforms. Typically, when a buffer comprising an acid is used in a liquidpharmaceutical composition, it is prepared using a salt form of the acidor a combination of the acid and a salt form of the acid. Thus, forexample, the buffer is prepared using the acid with its counterion, suchas sodium, potassium, ammonium, calcium, or magnesium. Hence, asuccinate buffer generally consists of a salt of succinic acid, such assodium succinate, or a mixture of succinic acid and sodium succinate.Although the acid used as a buffering agent in the stabilized liquidpharmaceutical compositions of the invention can be the salt form of theacid or a mixture of the acid and its salt form, preferably the acidserving as a buffering agent is solely in its acid form. Acids suitablefor use in formulating the stabilized liquid polypeptide-containingcompositions of the present invention include, but are not limited to,succinic acid, citric acid, phosphoric acid, glutamic acid, maleic acid,malic acid, acetic acid, tartaric acid, and aspartic acid, morepreferably succinic acid and citric acid, most preferably succinic acid.

The liquid polypeptide-containing pharmaceutical compositions of theinvention are “stabilized” compositions. By “stabilized” is intended theliquid compositions have increased storage stability relative tocompositions prepared in the absence of the combination of an amino acidbase and a buffering agent as disclosed herein. This increased storagestability is observed in the liquid formulation, whether stored directlyin that form for later use, stored in a frozen state and thawed prior touse, or prepared in a dried form, such as a lyophilized, air-dried, orspray-dried form, for later reconstitution into a liquid form or otherform prior to use. Preferably, compositions of the invention are storeddirectly in their liquid form to take full advantage of the convenienceof having increased storage stability in the liquid form, ease ofadministration without reconstitution, and ability to supply theformulation in prefilled, ready-to-use syringes or as multidosepreparations if the formulation is compatible with bacteriostaticagents.

The compositions of the invention relate to the discovery that theaddition of the amino acid arginine, lysine, aspartic acid, or glutamicacid in its free base form or in its salt form in combination with anacid substantially free of its salt form, an acid in its salt form, or amixture of an acid and its salt form, results in a liquidpolypeptide-containing pharmaceutical composition that has increasedstorage stability relative to a liquid polypeptide-containingpharmaceutical composition prepared without the combination of these twocomponents. The increased storage stability of the composition isachieved through the influence of the amino acid on stability of thetherapeutically active polypeptide, more particularly its influence onpolypeptide aggregation during storage in liquid formulations.Furthermore, incorporation of an amino acid base as defined herein andan acid substantially free of its salt form within liquidpolypeptide-containing formulations results in liquid pharmaceuticalcompositions that are near isotonic without having to include additionalisotonizing agents, such as sodium chloride. By “near isotonic” isintended the liquid composition has an osmolarity of about 240 mmol/kgto about 360 mmol/kg, preferably about 240 to about 340 mmol/kg, morepreferably about 250 to about 330 mmol/kg, even more preferably about260 to about 320 mmol/kg, most preferably about 270 to about 310mmol/kg.

The amino acid base incorporated into the stabilized liquidpharmaceutical compositions of the invention protects thetherapeutically active polypeptide against aggregation, therebyincreasing stability of the polypeptide during storage of thecomposition. By “increasing stability” is intended that aggregateformation by the polypeptide during storage of the liquid pharmaceuticalcomposition is decreased relative to aggregate formation of thepolypeptide during storage in the absence of this particular stabilizingagent. Decreased aggregate formation with addition of amino acid baseoccurs in a concentration dependent manner. That is, increasingconcentrations of amino acid base lead to increased stability of apolypeptide in a liquid pharmaceutical composition when that polypeptidenormally exhibits aggregate formation during storage in a liquidformulation in the absence of the amino acid base. Determination of theamount of a particular amino acid base to be added to a liquidpharmaceutical composition to decrease aggregate formation therebyincreasing polypeptide stability, and thus increasing storage stabilityof the composition, can readily be determined for any particularpolypeptide of interest without undue experimentation using methodsgenerally known to one of skill in the art.

Thus, for example, the effect of a particular amino acid base onpolypeptide aggregation during storage in a liquid composition can bereadily determined by measuring the change in soluble polypeptide insolution over time. Amount of soluble polypeptide in solution can bequantified by a number of analytical assays adapted to detection of thepolypeptide of interest. Such assays include, for example, reverse phase(RP)-HPLC, size exclusion (SEC)-HPLC, and UV absorbance, as described inthe Examples below. Where a polypeptide of interest forms both solubleand insoluble aggregates during storage in liquid formulations, acombination of RP-HPLC and SEC-HPLC can be used to distinguish betweenthat portion of the soluble polypeptide that is present as solubleaggregates and that portion that is present in the nonaggregate,biologically active molecular form, as described in Example 1 below.

In the case of aggregation, an effective amount of amino acid base toincorporate within a polypeptide-containing liquid pharmaceuticalcomposition to obtain the stabilized pharmaceutical composition of theinvention would be viewed as an amount that resulted in decreasedaggregate formation over time, and hence greater retention of solublepolypeptide in solution in its nonaggregated, biologically activemolecular form. Thus, for example, where the polypeptide is a monomericprotein, such as the interleukin-2 (IL-2) or tissue factor pathwayinhibitor (TFPI) described in the Examples below, an effective amount ofstabilizing agent for use in preparing a stabilized composition of theinvention would be an amount that resulted in greater retention of IL-2or TFPI in its monomeric molecular form.

Increased storage stability of the stabilized liquidpolypeptide-containing compositions of the invention may also beassociated with the inhibitory effects of the amino acid base ondeamidation of glutamine and/or asparagine residues within thetherapeutically active polypeptide during storage. The effect of aparticular amino acid base on deamidation of these residues duringstorage in a liquid composition can readily be determined by monitoringthe amount of polypeptide present in its deamidated form over time.Methods for measuring molecular species, i.e., native or deamidated, ofa particular polypeptide present in solution phase are generally knownin the art. Such methods include chromatographic separation of themolecular species and identification using polypeptide molecular weightstandards, such as with RP-HPLC as described in the Examples below.

Use of the novel combination of an amino acid base buffered by an acidsubstantially free of its salt form to increase polypeptide stabilitywithin the stabilized liquid pharmaceutical compositions of theinvention provides advantages over, for example, the use of an aminoacid in a succinic acid/sodium succinate buffer system. This novelcombination allows for preparation of near isotonic formulations havinghigher concentrations of the stabilizing amino acid than can be achievedwith the use of a buffer system that is a mixture of an acid and itssalt form. The higher concentration of the stabilizing amino acid allowsfor even greater increases in polypeptide stability, and thus increasedstorage stability of the formulation.

For example, when succinic acid is used to buffer arginine base added toa liquid formulation comprising the protein interleukin-2 (IL-2) andhaving a pH optimum for that protein (pH 5.8), the concentration ofarginine can be increased to 230 mM while still maintaining isotonicityof the formulation. This results in a doubling of IL-2 storage shelflife at 50° C., which is a measure of protein stability. Although asimilar IL-2 storage shelf life can be achieved using the same arginineconcentration and succinic acid/sodium succinate as the buffering agent,arginine must be added in its acidic form to achieve a similar pH, andthe resulting formulation is hypertonic (see Example 1, Table 1).

Similarly, when citric acid is used to buffer arginine base added to aliquid formulation comprising the protein tissue factor pathwayinhibitor (TFPI) and having a pH suitable for that protein (pH 5.5), theconcentration of arginine can be increased to 300 mM while stillmaintaining isotonicity of the formulation. This results in nearly a 50%increase in TFPI storage shelf life at 50° C. Although a similar TFPIstorage shelf life can be achieved using the same arginine concentrationand citric acid/sodium citrate as the buffering agent, arginine mustagain be added in its acidic form to achieve a similar pH, and theresulting formulation is hypertonic (see Example 8, Table 18). Theability to use higher concentrations of an amino acid as the primarystabilizing agent eliminates the need for more traditional polypeptidestabilizers such as bovine serum albumin or human serum albumin, whichare less desirable stabilizing agents because of potential viralcontamination.

In addition, isotonicity of liquid pharmaceutical compositions isdesirable as it results in reduced pain upon administration andminimizes potential hemolytic effects associated with hypertonic orhypotonic compositions. Thus, the stabilized compositions of theinvention not only have increased storage stability, but also have theadded benefit of substantially reduced pain upon administration whencompared with formulations using other more traditional buffer systemsconsisting of an acid and a salt form of the acid. For example, in oneembodiment of the invention, the stabilized liquid pharmaceuticalcomposition when injected exhibits reduced pain associated with burningand stinging relative to injection of normal saline (see Example 7).

Having identified the advantages of preparing liquid polypeptidecompositions of the invention with an amino acid base as the primarystabilizing agent and an acid substantially free of its salt form as thebuffering agent, it is within skill in the art to determine, withoutundue experimentation, preferred concentrations of each of thesecomponents to be incorporated into a liquid pharmaceutical compositioncomprising a therapeutically active polypeptide of interest thatexhibits aggregate formation as described herein to achieve increasedpolypeptide stability during storage of that composition. Following theprotocols disclosed, for example, in Example 1 below, the skilledartisan may assess a range of desired concentrations of the amino acidbase and the various buffering acids for use in the liquidpharmaceutical compositions described herein. Preferably the amount ofamino acid base incorporated into the composition is within aconcentration range of about 100 mM to about 400 mM, preferably about130 mM to about 375 mM, more preferably about 150 mM to about 350 mM,even more preferably about 175 mM to about 325 mM, still more preferablyabout 180 mM to about 300 mM, even more preferably about 190 mM to about280 mM, most preferably about 200 mM to about 260 mM, depending upon theprotein present in the composition. Although the buffering agent may bethe acid in its salt form, or a mixture of the acid and its salt form,preferably the buffering agent is the acid substantially free of itssalt form, for the advantageous reasons disclosed herein. The acid usedas the buffering agent is preferably added within a concentration rangeof about 40 mM to about 250 mM, about 50 mM to about 240 mM, about 60 mMto about 230 mM, about 70 mM to about 220 mM, more preferably about 80mM to about 210 mM, most preferably about 90 mM to about 200 mM,depending upon the acid used as the buffering agent and the pH optimumfor the polypeptide being stabilized against aggregate formation.

In one embodiment, the amino acid base is arginine base present at aconcentration of about 230 mM and the acid used as the buffering agentis succinic acid at a concentration of about 128 mM. This allows for thepreparation of a liquid polypeptide-containing pharmaceuticalcomposition having an osmolarity that is near isotonic and a pH of about5.8. In another embodiment, the amino acid base is arginine base presentat a concentration of about 300 mM and the acid used as the bufferingagent is citric acid at a concentration of about 120 mM. This allows forthe preparation of a liquid polypeptide-containing pharmaceuticalcomposition having an osmolarity that is near isotonic and a pH of about5.5. In yet another embodiment, the amino acid base is arginine basepresent at a concentration of about 200 mM to about 300 mM and the acidused as the buffering agent is succinic acid at a concentration of about120 mM to about 180 mM. This allows for the preparation of a liquidpolypeptide-containing pharmaceutical composition having an osmolarityof about 256 mmol/kg to about 363 mmol/kg and a pH of about 5.5.

Thus, in another embodiment of the invention, the stabilized liquidpharmaceutical composition comprises IL-2 or variant thereof as thepolypeptide, arginine base at a concentration of about 150 mM to about350 mM, and succinic acid at a concentration of about 80 mM to about 190mM. In a preferred embodiment, the arginine base is present in the IL-2liquid pharmaceutical composition at a concentration of about 230 mM andsuccinic acid is present at a concentration of about 128 mM. Thispreferred IL-2 composition has a pH of about 5.8 and an osmolarity ofabout 250 mmol/kg to about 330 mmol/kg. The concentration of 12 orvariant thereof in these compositions is about 0.01 mg/ml to about 2.0mg/ml, preferably about 0.02 mg/ml to about 1.0 mg/ml, more preferablyabout 0.03 mg/ml to about 0.8 mg/ml, most preferably about 0.03 mg/ml toabout 0.5 mg/ml.

In yet another embodiment of the invention, the stabilized liquidpharmaceutical composition comprises TFPI or variant thereof as thepolypeptide, arginine base at a concentration of about 100 mM to about400 mM, and succinic acid at a concentration of about 80 mM to about 190mM. In a preferred embodiment, the arginine base is present in the TFPIliquid pharmaceutical composition at a concentration of about 200 mM toabout 300 mM and succinic acid is present at a concentration of about120 mM to about 180 mM. This preferred TFPI composition has a pH ofabout 5.5 and an osmolarity of about 240 mmol/kg to about 360 mmol/kg.The concentration of TFPI or variant thereof in these compositions isabout 0.01 mg/ml to about 5.0 mg/ml, preferably about 0.05 mg/ml toabout 2.0 mg/ml, more preferably about 0.10 mg/ml to about 1.0 mg/ml,most preferably about 0.10 mg/ml to about 0.60 mg/ml.

In another embodiment of the invention, the stabilized liquidpharmaceutical composition comprises TFPI or variant thereof as thepolypeptide, arginine base at a concentration of about 175 mM to about400, and citric acid at a concentration of about 40 mM to about 200 mM.In a preferred embodiment, the arginine base is present in the TFPIliquid pharmaceutical composition at a concentration of about 250 mM toabout 350 mM and citric acid is present at a concentration of about 100mM to about 150 mM. This preferred TFPI composition has a pH of about5.0-6.5 and an osmolarity of about 240 mmol/kg to about 360 mmol/kg. Inyet another embodiment, the arginine base is present at a concentrationof about 300 mM and citric acid is present at a concentration of about120 mM. This TFPI composition has a pH of about 5.5 and an osmolarity ofabout 240 mmol/kg to about 360 mmol/kg. The concentration of TFPI orvariant thereof in these compositions is about 0.01 mg/ml to about 5.0mg/ml, preferably about 0.05 mg/ml to about 2.0 mg/ml, more preferablyabout 0.10 mg/ml to about 1.0 mg/ml, most preferably about 0.10 mg/ml toabout 0.60 mg/ml.

As shown in the examples below, pH of a liquid polypeptide-containingpharmaceutical formulation affects the stability of the polypeptidecontained therein, primarily through its affect on polypeptide aggregateformation. Thus the amount of buffering acid present in thepharmaceutical compositions of the invention will vary depending uponthe pH optimum for stability of a particular polypeptide of interest.Determination of this pH optimum can be achieved using methods generallyavailable in the art, and further illustrated in the Examples describedherein. Preferred pH ranges for the compositions of the invention areabout pH 4.0 to about pH 9.0, more particularly about pH 5.0 to about6.5, depending upon the polypeptide. Thus, in one embodiment, the pH isabout 5.8, more particularly when the polypeptide is 12 or variantthereof. In another embodiment, the pH is about 5.5, more particularlywhen the polypeptide is TFPI or variant thereof.

The stabilized pharmaceutical compositions comprising an amino acid basebuffered with an acid substantially free of its salt form, the salt formof the acid, or a mixture of the acid and its salt form, may alsocomprise additional stabilizing agents, which further enhance stabilityof a therapeutically active polypeptide therein. Stabilizing agents ofparticular interest to the present invention include, but are notlimited to, methionine and EDTA, which protect the polypeptide againstmethionine oxidation; and a nonionic surfactant, which protects thepolypeptide against aggregation associated with freeze-thawing ormechanical shearing.

In this manner, the amino acid methionine may be added to inhibitoxidation of methionine residues to methionine sulfoxide when thepolypeptide acting as the therapeutic agent is a polypeptide comprisingat least one methionine residue susceptible to such oxidation. By“inhibit” is intended minimal accumulation of methionine oxidizedspecies over time. Inhibiting methionine oxidation results in greaterretention of the polypeptide in its proper molecular form. Anystereoisomer of methionine (L, D, or DL isomer) or combinations thereofcan be used. The amount to be added should be an amount sufficient toinhibit oxidation of the methionine residues such that the amount ofmethionine sulfoxide is acceptable to regulatory agencies. Typically,this means that the composition contains no more than about 10% to about30% methionine sulfoxide. Generally, this can be achieved by addingmethionine such that the ratio of methionine added to methionineresidues ranges from about 1:1 to about 1000:1, most preferably 10:1 toabout 100:1.

The preferred amount of methionine to be added can readily be determinedempirically by preparing the composition comprising the polypeptide ofinterest with different concentrations of methionine and determining therelative effect on formation of oxidative species of the polypeptideusing, for instance, chromatographic separation of the molecular speciesand identification using polypeptide molecular weight standards, such aswith RP-HPLC, as described below in Example 2. That concentration ofmethionine that maximizes inhibition of oxidation of methionineresidues, without having adverse affects on amino acid-relatedinhibition of polypeptide aggregation, would represent a preferredamount of methionine to be added to the composition to further improvepolypeptide stability.

Polypeptide degradation due to freeze thawing or mechanical shearingduring processing of the liquid composition of the present invention canbe inhibited by incorporation of surfactants into the liquidpolypeptide-containing compositions of the invention in order to lowerthe surface tension at the solution-air interface. Typical surfactantsemployed are nonionic surfactants, including polyoxyethylene sorbitolesters such as polysorbate 80 (TWEEN 80®) and polysorbate 20 (TWEEN20®); polyoxypropylene-polyoxyethylene esters such as PLURONIC F68®;polyoxyethylene alcohols such as Brij 35®; simethicone; polyethyleneglycol such as PEG400; lysophosphatidylcholine; andpolyoxyethylene-p-t-octylphenol such as Triton X-100®. Classicstabilization of pharmaceuticals by surfactants or emulsifiers isdescribed, for example, in Levine et al. (1991) J. Parenteral Sci.Technol. 45(3):160-165, herein by reference. A preferred surfactantemployed in the practice of the present invention is polysorbate 80.

In addition to those agents disclosed above, other stabilizing agents,such as albumin, ethylenediaminetetracetic acid (EDTA) or one of itssalts such as disodium EDTA, can be added to further enhance thestability of the liquid pharmaceutical compositions. The amount ofalbumin can be added at concentrations of about 1.0% w/v or less. TheEDTA acts as a scavenger of metal ions known to catalyze many oxidationreactions, thus providing an additional stabilizing agent.

In one embodiment of the invention, the stabilized liquid pharmaceuticalcomposition comprises IL-2 or variant thereof as the polypeptide,arginine base at a concentration of about 150 mM to about 350 mM,succinic acid at a concentration of about 80 mM to about 190 mM,methionine at a concentration of about 0.5 mM to about 10 mM, EDTA atabout 0.1 to about 5.0 mM, and polysorbate 80 at about 0.001% to about0.2%. In a preferred embodiment, the arginine base is present in thisIL-2 liquid pharmaceutical composition at a concentration of about 230mM and succinic acid is present at a concentration of about 128 mM. Thispreferred IL-2 composition has a pH of about 5.8 and an osmolarity ofabout 250 mmol/kg to about 330 mmol/kg. The concentration of IL-2 orvariant thereof in these compositions is about 0.01 mg/ml to about 2.0mg/ml, preferably about 0.02 mg/ml to about 1.0 mg/ml, more preferablyabout 0.03 mg/ml to about 0.8 mg/ml, most preferably about 0.03 mg/ml toabout 0.5 mg/ml.

Where desirable, sugars or sugar alcohols may also be included in thestabilized liquid polypeptide-containing pharmaceutical compositions ofthe present invention. Any sugar such as mono-, di-, or polysaccharides,or water-soluble glucans, including for example fructose, glucose,mannose, sorbose, xylose, maltose, lactose, sucrose, dextran, pullulan,dextrin, cyclodextrin, soluble starch, hydroxyethyl starch andcarboxymethylcellulose-Na may be used. Sucrose is the most preferredsugar additive.

Sugar alcohol is defined as a C4-C8 hydrocarbon having an —OH group andincludes, for example, mannitol, sorbitol, inositol, galacititol,dulcitol, xylitol, and arabitolm with mannitol being the most preferredsugar alcohol additive. The sugars or sugar alcohols mentioned above maybe used individually or in combination. There is no fixed limit to theamount used, as long as the sugar or sugar alcohol is soluble in theliquid preparation and does not adversely effect the stabilizing effectsachieved using the methods of the invention. Preferably, the sugar orsugar alcohol concentration is between about 1.0 w/v % and about 15.0w/v %, more preferably between about 2.0 w/v % and about 10.0 w/v %.

The stabilized liquid pharmaceutical compositions of the invention maycontain other compounds that increase the effectiveness or promote thedesirable qualities of the polypeptide of interest that serves as atherapeutically active component so long as the primary stabilizingeffect achieved with the amino acid base is not adversely affected. Thecomposition must be safe for administration via the route that ischosen, it must it must be sterile, and must retain its desiredtherapeutic activity.

Compositions of the present invention are preferably prepared bypremixing the stabilizing and buffering agents, and any other excipientsprior to incorporation of the polypeptide of interest. Any additionalexcipients that may be added to further stabilize the compositions ofthe present invention must not adversely affect the stabilizing effectsof the primary stabilizing agent, i.e., an amino acid base, incombination with the buffering agent, i.e., an acid substantially freeof its salt form, the salt form of the acid, or a mixture of the acidand its salt form, as used to obtain the novel compositions disclosedherein. Following addition of a preferred amount of an amino acid baseto achieve decreased aggregate formation of a polypeptide of interest,pH of the liquid composition is adjusted using the buffering agent,preferably within a range disclosed herein, more preferably to the pHoptimum for the polypeptide of interest. Although pH can be adjustedfollowing addition of the polypeptide of interest into the composition,preferably it is adjusted prior to addition of this polypeptide, as thiscan reduce the risk of denaturation the polypeptide. Appropriatemechanical devices are then used for achieving a proper mix ofconstituents.

While specific embodiments of the invention are directed to stabilizedcompositions comprising interleukin-2 (IL-2) or variant thereof, ortissue factor pathway inhibitor (TFPI) or variant thereof, examples ofproteins that are particularly susceptible to degradation via aggregateformation, the utility of the invention extends generally to anypharmaceutical composition containing a polypeptide or variant thereofthat exhibits aggregate formation during storage in a liquidformulation. Thus polypeptides suitable for use in the practice of thepresent invention include; for example, interleukins (e.g., IL-2),interferons including β-interferon (IFN-β) and its muteins such asIFN-β_(ser17) (as described in European Patent Application No. 185459B1and U.S. Pat. No. 4,588,585, incorporated herein by reference), tissuefactor pathway inhibitor (TFPI), human growth hormone (hGH), insulin,and other like polypeptides that exhibit aggregate formation in a liquidformulation, as well as any biologically active variants thereof.

The polypeptides present in the stabilized liquid pharmaceuticalcompositions of the invention may be native or obtained by recombinanttechniques, and may be from any source, including mammalian sources suchas, e.g., mouse, rat, rabbit, primate, pig, and human, provided theymeet the criterion specified herein, that is, provided they formaggregates during storage in liquid formulations. Preferably suchpolypeptides are derived from a human source, and more preferably arerecombinant, human proteins from microbial hosts.

Biologically active variants of a polypeptide of interest that serves asa therapeutically active component in the pharmaceutical compositions ofthe invention are also encompassed by the term “polypeptide” as usedherein. Such variants should retain the desired biological activity ofthe native polypeptide such that the pharmaceutical compositioncomprising the variant polypeptide has the same therapeutic effect asthe pharmaceutical composition comprising the native polypeptide whenadministered to a subject. That is, the variant polypeptide will serveas a therapeutically active component in the pharmaceutical compositionin a manner similar to that observed for the native polypeptide. Methodsare available in the art for determining whether a variant polypeptideretains the desired biological activity, and hence serves as atherapeutically active component in the pharmaceutical composition.Biological activity can be measured using assays specifically designedfor measuring activity of the native polypeptide or protein, includingassays described in the present invention. Additionally, antibodiesraised against a biologically active native polypeptide can be testedfor their ability to bind to the variant polypeptide, where effectivebinding is indicative of a polypeptide having a conformation similar tothat of the native polypeptide.

Suitable biologically active variants of a native or naturally occurringpolypeptide of interest can be fragments, analogues, and derivatives ofthat polypeptide. By “fragment” is intended a polypeptide consisting ofonly a part of the intact polypeptide sequence and structure, and can bea C-terminal deletion or N-terminal deletion of the native polypeptide.By “analogue” is intended an analogue of either the native polypeptideor of a fragment of the native polypeptide, where the analogue comprisesa native polypeptide sequence and structure having one or more aminoacid substitutions, insertions, or deletions. “Muteins”, such as thosedescribed herein, and peptides having one or more peptoids (peptidemimics) are also encompassed by the term analogue (see InternationalPublication No. WO 91/04282). By “derivative” is intended any suitablemodification of the native polypeptide of interest, of a fragment of thenative polypeptide, or of their respective analogues, such asglycosylation, phosphorylation; or other addition of foreign moieties,so long as the desired biological activity of the native polypeptide isretained. Methods for making polypeptide fragments, analogues, andderivatives are generally available in the art.

For example, amino acid sequence variants of the polypeptide can beprepared by mutations in the cloned DNA sequence encoding the nativepolypeptide of interest. Methods for mutagenesis and nucleotide sequencealterations are well known in the art. See, for example, Walker andGaastra, eds. (1983) Techniques in Molecular Biology (MacMillanPublishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA82:488-492; Kunkel et al. (1987) Methods Enzymol. 154:367-382; Sambrooket al. (1989) Molecular Cloning. A Laboratory Manual (Cold SpringHarbor, N.Y.); U.S. Pat. No. 4,873,192; and the references citedtherein; herein incorporated by reference. Guidance as to appropriateamino acid substitutions that do not affect biological activity of thepolypeptide of interest may be found in the model of Dayhoff et al.(1978) in Atlas of Protein Sequence and Structure (Natl. Biomed. Res.Found., Washington, D.C.), herein incorporated by reference.Conservative substitutions, such as exchanging one amino acid withanother having similar properties, may be preferred. Examples ofconservative substitutions include, but are not limited to, Gly

Ala, Val

Ile

Leu, Asp

Glu, Lys

Arg, Asn

Gln, and Phe

Trp

Tyr.

In constructing variants of the polypeptide of interest, modificationsare made such that variants continue to possess the desired activity.Obviously, any mutations made in the DNA encoding the variantpolypeptide must not place the sequence out of reading frame andpreferably will not create complementary regions that could producesecondary mRNA structure. See EP Patent Application Publication No.75,444.

Biologically active variants of a polypeptide of interest will generallyhave at least 70%, preferably at least 80%, more preferably about 90% to95% or more, and most preferably about 98% or more amino acid sequenceidentity to the amino acid sequence of the reference polypeptidemolecule, which serves as the basis for comparison. A biologicallyactive variant of a native polypeptide of interest may differ from thenative polypeptide by as few as 1-15 amino acids, as few as 1-10, suchas 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.By “sequence identity” is intended the same amino acid residues arefound within the variant polypeptide and the polypeptide molecule thatserves as a reference when a specified, contiguous segment of the aminoacid sequence of the variant is aligned and compared to the amino acidsequence of the reference molecule. The percentage sequence identitybetween two amino acid sequences is calculated by determining the numberof positions at which the identical amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the segmentundergoing comparison to the reference molecule, and multiplying theresult by 100 to yield the percentage of sequence identity.

For purposes of optimal alignment of the two sequences, the contiguoussegment of the amino acid sequence of the variant may have additionalamino acid residues or deleted amino acid residues with respect to theamino acid sequence of the reference molecule. The contiguous segmentused for comparison to the reference amino acid sequence will compriseat least twenty (20) contiguous amino acid residues, and may be 30, 40,50, 100, or more residues. Corrections for increased sequence identityassociated with inclusion of gaps in the variant's amino acid sequencecan be made by assigning gap penalties. Methods of sequence alignmentare well known in the art for both amino acid sequences and for thenucleotide sequences encoding amino acid sequences.

Thus, the determination of percent identity between any two sequencescan be accomplished using a mathematical algorithm. One preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller (1988)CABIOS 4:11-17. Such an algorithm is utilized in the ALIGN program(version 2.0), which is part of the GCG sequence alignment softwarepackage. A PAM120 weight residue table, a gap length penalty of 12, anda gap penalty of 4 can be used with the ALIGN program when comparingamino acid sequences. Another preferred, nonlimiting example of amathematical algorithm for use in comparing two sequences is thealgorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLASTand XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403.BLAST nucleotide searches can be performed with the NBLAST program,score=100, wordlength=12, to obtain nucleotide sequences homologous to anucleotide sequence encoding the polypeptide of interest. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3, to obtain amino acid sequences homologous to thepolypeptide of interest. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be usedto perform an iterated search that detects distant relationships betweenmolecules. See Altschul et al. (1997) supra. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Also see the ALIGN program (Dayhoff (1978)in Atlas of Protein Sequence and Structure 5:Suppl. 3 (NationalBiomedical Research Foundation, Washington, D.C.) and programs in theWisconsin Sequence Analysis Package, Version 8 (available from GeneticsComputer Group, Madison, Wis.), for example, the GAP program, wheredefault parameters of the programs are utilized.

When considering percentage of amino acid sequence identity, some aminoacid residue positions may differ as a result of conservative amino acidsubstitutions, which do not affect properties of protein function. Inthese instances, percent sequence identity may be adjusted upwards toaccount for the similarity in conservatively substituted amino acids.Such adjustments are well known in the art. See, for example, Myers andMiller (1988) Computer Applic. Biol. Sci. 4:11-17.

The precise chemical structure of a polypeptide depends on a number offactors. As ionizable amino and carboxyl groups are present in themolecule, a particular polypeptide may be obtained as an acidic or basicsalt, or in neutral form. All such preparations that retain theirbiological activity when placed in suitable environmental conditions areincluded in the definition of polypeptides as used herein. Further, theprimary amino acid sequence of the polypeptide may be augmented byderivatization using sugar moieties (glycosylation) or by othersupplementary molecules such as lipids, phosphate, acetyl groups and thelike. It may also be augmented by conjugation with saccharides. Certainaspects of such augmentation are accomplished through post-translationalprocessing systems of the producing host; other such modifications maybe introduced in vitro. In any event, such modifications are included inthe definition of polypeptide used herein so long as the activity of thepolypeptide is not destroyed. It is expected that such modifications mayquantitatively or qualitatively affect the activity, either by enhancingor diminishing the activity of the polypeptide, in the various assays.Further, individual amino acid residues in the chain may be modified byoxidation, reduction, or other derivatization, and the polypeptide maybe cleaved to obtain fragments that retain activity. Such alterationsthat do not destroy activity do not remove the polypeptide sequence fromthe definition of polypeptide of interest as used herein.

The art provides substantial guidance regarding the preparation and useof polypeptide variants. In preparing the polypeptide variants, one ofskid in the art can readily determine which modifications to the nativeprotein nucleotide or amino acid sequence will result in a variant thatis suitable for use as a therapeutically active component of apharmaceutical composition of the present invention and whose aggregateformation is decreased by the presence of an amino acid base and an acidsubstantially free of its salt form, the salt form of the acid, or amixture of the acid and its salt form, as described herein.

In one embodiment of the invention, the polypeptide present as atherapeutically active component in the liquid pharmaceuticalcomposition of the invention is interleukin-2 (IL-2) or variant thereof,preferably recombinant IL-2. Interleukin-2 is a lymphokine that isproduced by normal peripheral blood lymphocytes and is present in thebody at low concentrations. It induces the proliferation of antigen- ormitogen-stimulated T cells after exposure to plant lectins, antigens, orother stimuli. IL-2 was first described by Morgan et al. (1976) Science193:1007-1008 and originally called T-cell growth factor because of itsability to induce proliferation of stimulated T lymphocytes. It is aprotein with a reported molecular weight in the range of 13,000 to17,000 (Gillis and Watson (1980) J. Exp. Med. 159:1709) and has anisoelectric point in the range of 6-8.5. It is now recognized that inaddition to its growth factor properties, it modulates various in vitroand in vivo functions of the immune system. IL-2 is one of severallymphocyte-produced messenger-regulatory molecules that mediate cellularinteractions and functions. This naturally occurring lymphokine has beenshown to have antitumor activity against a variety of malignancieseither alone or when combined with lymphokine-activated killer (LAK)cells or tumor-infiltrating lymphocytes (see, for example, Rosenberg etal. (1987) N. Engl. J. Med. 316:889-897; Rosenberg (1988) Ann. Surg.208:121-135; Topalian et al. 1988) J. Clin. Oncol. 6:839-853; Rosenberget al. (1988) N. Engl. J. Med. 319:1676-1680; and Weber et al. (1992) J.Clin. Oncol. 10:33-40). Although the anti-tumor activity of IL-2 hasbest been described in patients with metastatic melanoma and renal cellcarcinoma, other diseases, notably lymphoma, also appear to respond totreatment with IL-2.

By “recombinant IL-2” is intended interleukin-2 having comparablebiological activity to native-sequence IL-2 and which has been preparedby recombinant DNA techniques as described, for example, by Taniguchi etal. (1983) Nature 302:305-310 and Devos (1983) Nucleic Acids Research11:4307-4323 or mutationally altered IL-2 as described by Wang et al.(1984) Science 224:1431-1433. In general, the gene coding for IL-2 iscloned and then expressed in transformed organisms, preferably amicroorganism, and most preferably E. coli, as described herein. Thehost organism expresses the foreign gene to produce IL-2 underexpression conditions. Synthetic recombinant IL-2 can also be made ineukaryotes, such as yeast or human cells. Processes for growing,harvesting, disrupting, or extracting the IL-2 from cells aresubstantially described in, for example, U.S. Pat. Nos. 4,604,377;4,738,927; 4,656,132; 4,569,790; 4,748,234; 4,530,787; 4,572,298; and4,931,543; herein incorporated by reference in their entireties.

For examples of variant IL-2 proteins, see European Patent ApplicationNo. 136,489; European Patent Application No. 83101035.0 filed Feb. 3,1983 (published Oct. 19, 1983 under Publication No. 91539); EuropeanPatent Application No. 82307036.2, filed Dec. 22, 1982 (published Sep.14, 1983 under No. 88195); the recombinant IL-2 muteins described inEuropean Patent Application No. 83306221.9, filed Oct. 13, 1983(published May 30, 1984 under No. 109748), which is the equivalent toBelgian Patent No. 893,016, commonly owned U.S. Pat. No. 4,518,584; themuteins described in U.S. Pat. No. 4,752,585 and WO 99/60128; and theIL-2 mutein used in the examples herein and described in U.S. Pat. No.4,931,543; all of which are herein incorporated by reference.Additionally, IL-2 can be modified with polyethylene glycol to provideenhanced solubility and an altered pharmokinetic profile (see U.S. Pat.No. 4,766,106, hereby incorporated by reference in its entirety).

In another embodiment of the invention, the polypeptide present as atherapeutically active component in the liquid pharmaceuticalcomposition of the invention is an interferon, more particularly thefibroepithelial β-interferon (IFN-β) or variant thereof, preferablyrecombinant IFN-β prepared by recombinant DNA techniques described inthe art. Interferons are produced by mammalian cells in response toexposure to a variety of inducers, such as mitogens, polypeptides,viruses, and the like. These relatively small, species-specific, singlechain polypeptides exhibit immunoregulatory, antiviral, andantiproilferative properties. Interferons are of interest as therapeuticagents for treatment of antiviral diseases and control of cancer.

DNA sequences encoding the human IFN-β gene are available in the art(see Goeddel et al (1980) Nucleic Acids Res. 8:4057 and Taniguchi et al(1979) Proc. Japan acad. Sci. 855:464) and the gene has been expressedin E. coli (Taniguchi et al. (1980) Gene 10:11-15) and Chinese hamsterovary cells (see, for example, U.S. Pat. Nos. 4,966,843 and 5,376,567).Variants of IFN-β are described in European Patent Application No.185459B1, and U.S. Pat. Nos. 4,518,584, 4,588,585, and 4,737,462describe muteins such as IFN-β_(ser17) expressed in E. coli, all ofwhich are herein incorporated by reference.

In yet another embodiment, the polypeptide present as a therapeuticallyactive component in the liquid pharmaceutical composition of theinvention is tissue factor pathway inhibitor (TFPI) or variant thereof,preferably recombinant TFPI. This polypeptide, which is an inhibitor ofthe coagulation cascade, is also known as lipoprotein associatedcoagulation inhibitor (LACI), tissue factor inhibitor (TFI), andextrinsic pathway inhibitor (EPI). TFPI was first purified from a humanhepatoma cell, Hep G2 (Broze and Miletich (1987) Proc. Natl. Acad. Sci.USA 84:1886-1890) and subsequently from human plasma (Novotny et al.(1989) J. Biol. Chem. 264:18832-18837); and Chang liver and SK hepatomacells (Wun et al. (1990) J. Biol. Chem. 255:16096-16101). TFPI cDNA havebeen isolated from placental and endothelial cDNA libraries (Wun et al.(1988) J. Biol. Chem. 263:6001-6004); Girard et al. (1989) Thromb. Res.55:37-50). For reviews, see Rapaport (1989) Blood 73:359-365 (1989);Broze et al. (1990) Biochemistry 29:7539-7546. The cloning of the TFPIcDNA, which encodes the 276 amino acid residue protein of TFPI, isfurther described in U.S. Pat. No. 4,966,852; see also U.S. Pat. Nos.5,773,251 and 5,849,875; all of which are herein incorporated byreference.

Variants of TFPI are known in the art. See, for example, U.S. Pat. No.5,212,091, where a non-glycosylated form of recombinant TFPI has beenproduced and isolated from E. coli; U.S. Pat. No. 5,106,833, whereanalogues and fragments are disclosed; and U.S. Pat. No. 5,378,614,where production of TFPI analogues in yeast is described; all of whichare herein incorporated by reference. Also, the above-mentioned U.S.Pat. No. 5,849,875 describes the 276 amino acid residue polypeptideconsisting of mature tissue factor inhibitor, immediately preceded by anN-terminal methionine residue, alanine residue, or methionine-alaninedi-peptide.

A pharmaceutically effective amount of a stabilizedpolypeptide-containing liquid pharmaceutical composition of theinvention is administered to a subject. By “pharmaceutically effectiveamount” is intended an amount that is useful in the treatment,prevention or diagnosis of a disease or condition. Typical routes ofadministration include, but are not limited to, oral administration andparenteral administration, including intravenous, intramuscular,subcutaneous, intraarterial and intraperitoneal injection or infusion.In one such embodiment, the administration is by injection, preferablysubcutaneous injection. Injectable forms of the compositions of theinvention include, but are not limited to, solutions, suspensions andemulsions.

The stabilized liquid pharmaceutical composition comprising thepolypeptide of interest should be formulated in a unit dosage and may bein an injectable or infusible form such as solution, suspension, oremulsion. Furthermore, it can be stored frozen or prepared in the driedform, such as a lyophilized powder, which can be reconstituted into theliquid solution, suspension, or emulsion before administration by any ofvarious methods including oral or parenteral routes of administration.Preferably it is stored in the liquid formulation to take advantage ofthe increased storage stability achieved in accordance with the methodsof the present invention as outlined below. The stabilizedpharmaceutical composition is preferably sterilized by membranefiltration and is stored in unit-dose or multi-dose containers such assealed vials or ampules. Additional methods for formulating apharmaceutical composition generally known the art may be used tofurther enhance storage stability of the liquid pharmaceuticalcompositions disclosed herein provided they do not adversely affect thebeneficial effects of the preferred stabilizing and buffering agentsdisclosed in the methods of the invention. A thorough discussion offormulation and selection of pharmaceutically acceptable carriers,stabilizers, etc. can be found in Remington's Pharmaceutical Sciences(1990) (18^(th) ed., Mack Pub. Co., Eaton, Pa.), herein incorporated byreference.

By “subject” is intended any animal. Preferably the subject ismammalian, must preferably the subject is human. Mammals of particularimportance other than human include, but are not limited to, dogs, cats,cows, horses, sheep, and pigs.

When administration is for the purpose of treatment, administration maybe for either a prophylactic or therapeutic purpose. When providedprophylactically, the substance is provided in advance of any symptom.The prophylactic administration of the substance serves to prevent orattenuate any subsequent symptom. When provided therapeutically, thesubstance is provided at (or shortly after) the onset of a symptom. Thetherapeutic administration of the substance serves to attenuate anyactual symptom.

Thus, for example, formulations comprising an effective amount of apharmaceutical composition of the invention comprising native-sequenceIL-2 or variant thereof can be used for the purpose of treatment,prevention, and diagnosis of a number of clinical indications responsiveto therapy with this polypeptide. Biologically active variants ofnative-sequence IL-2, such as muteins of IL-2 that retain IL-2 activity,in particular the mutein IL-2.sub.ser125 and other muteins in which thecysteine at position 125 has been replaced with another amino acid, canbe formulated and used in the same manner as native-sequence IL-2.Accordingly, formulations of the invention comprising native-sequenceIL-2 or variant thereof are useful for the diagnosis, prevention, andtreatment (local or systemic) of bacterial, viral, parasitic, protozoanand fungal infections; for augmenting cell-mediated cytotoxicity; forstimulating lymphokine activated killer (LAK) cell activity; formediating recovery of immune function of lymphocytes; for augmentingalloantigen responsiveness; for facilitating recovery of immune functionin acquired immune deficient states; for reconstitution of normalimmunofunction in aged humans and animals; in the development ofdiagnostic assays such as those employing enzyme amplification,radiolabelling, radioimaging, and other methods known in the art formonitoring IL-2 levels in the diseased state; for the promotion ofT-cell growth in vitro for therapeutic and diagnostic purposes; forblocking receptor sites for lymphokines; and in various othertherapeutic, diagnostic and research applications. The varioustherapeutic and diagnostic applications of human IL-2 or variantsthereof, such as IL-2 muteins, have been investigated and reported inRosenberg et al. (1987) N. Engl. J. Med. 316:889-897; Rosenberg (1988)Ann. Surg. 208:121-135; Topalian et al. 1988) J. Clin. Oncol. 6:839-853;Rosenberg et al. (1988) N. Engl. J. Med. 319:1676-1680; Weber et al.(1992) J. Clin. Oncol. 10:33-40; Grimm et al. (1982) Cell. Immunol.70(2):248-259; Mazumder (1997) Cancer J. Sci. Am. 3(Suppl. 1):S37-42;Mazumder and Rosenberg (1984) J. Exp. Med. 159(2):495-507; and Mazumderet al. (1983) Cancer Immunol. Immunother. 15(1):1-10. Formulations ofthe invention comprising IL-2 or variant thereof may be used as thesingle therapeutically active agent or may be used in combination withother immunologically relevant B or T cells or other therapeutic agents.Examples of relevant cells are B or T cells, natural killer cells, LAKcells, and the like, and exemplary therapeutic reagents that may be usedin combination with IL-2 or variant thereof are the various interferons,especially gamma interferon, B-cell growth factor, IL-1, and antibodies,for example anti-HER2 or anti-CD20 antibodies. Formulations of theinvention comprising IL-2 or variant thereof may be administered tohumans or animals orally, intraperitoneally, intramuscularly,subcutaneously, intravenously, intranasally, or by pulmonary delivery asdeemed appropriate by the physician. The amount of IL-2 (eithernative-sequence or variant thereof retaining IL-2 biological activity,such as muteins disclosed herein) administered may range between about0.1 to about 15 mIU/m². For indications such as renal cell carcinoma andmetastatic melanoma, the IL-2 or biologically active variant thereof maybe administered as a high-dose intravenous bolus at 300,000 to 800,000IU/kg/8 hours.

Formulations comprising an effective amount of the pharmaceuticalcompositions of the invention comprising native-sequence tissue factorpathway inhibitor (TFPI) or variant thereof are useful for thediagnosis, prevention, and treatment (local or systemic) of clinicalindications responsive to therapy with this polypeptide. Such clinicalindications include, for example, indications associated with increasedsynthesis and release of neutrophil elastase, such as inflammatorydiseases including severe acute pancreatitis, emphysema, rheumatoidarthritis, multiple organ failure, cystic fibrosis, Adult RespiratoryDistress Syndrome (ARDS), and sepsis; and for the diagnosis andtreatment of diseases associated with increased synthesis and release ofIL-8, including inflammatory diseases such as ARDS, reperfusion injury(including lung reperfusion injury), sepsis, and arthritis. See WO96/40224, herein incorporated by reference. Administration of IFN-β orits muteins to humans or animals may be delivered orally,intraperitoneally, intramuscularly, subcutaneously, intravenously,intranasally, or by pulmonary delivery as deemed appropriate by thephysician.

Formulations comprising an effective amount of the pharmaceuticalcompositions of the invention comprising β-interferon (IFN-β) or variantthereof, such as IFN-β_(ser17), are useful in the diagnosis, prevention,and treatment (local or systemic) of clinical indications responsive totherapy with this polypeptide. Such clinical indications include, forexample, disorders or diseases of the central nervous system (CNS),brain, and/or spinal cord, including Alzheimer's disease, Parkinson'sdisease, Lewy body dementia, multiple sclerosis, epilepsy, cerebellarataxia, progressive supranuclear palsy, amyotrophic lateral sclerosis,affective disorders, anxiety disorders, obsessive compulsive disorders,personality disorders, attention deficit disorder, attention deficithyperactivity disorder, Tourette Syndrome, Tay Sachs, Nieman Pick, andschizophrenia; nerve damage from cerebrovascular disorders such asstroke in the brain or spinal cord, from CNS infections includingmeningitis and HIV, from tumors of the brain and spinal cord, or from aprion disease; autoimmune diseases, including acquired immunedeficiency, rheumatoid arthritis, psoriasis, Crohn's disease, Sjogren'ssyndrome, amyotropic lateral sclerosis, and lupus; and cancers,including breast, prostate, bladder, kidney and colon cancers.Administration of IFN-β or its muteins to humans or animals may bedelivered orally, intraperitoneally, intramuscularly, subcutaneously,intravenously, intranasally, or by pulmonary delivery as deemedappropriate by the physician.

The present invention also provides a method for increasing stability ofa polypeptide in a liquid pharmaceutical composition, where thepolypeptide, which serves as a therapeutically active component,exhibits aggregate formation during storage in a liquid formulation. Themethod comprises incorporating into the liquid pharmaceuticalcomposition an amino acid base in an amount sufficient to decreaseaggregate formation of the polypeptide during storage of the liquidpharmaceutical composition, and an acid substantially free of its saltform, an acid in its salt form, or a mixture of an acid and its saltform, where the acid serves as a buffering agent to maintain the pH ofthe liquid composition within an acceptable range, as previouslydescribed herein.

Increasing stability of a polypeptide or variant thereof byincorporating an amino acid base, or an amino acid base base plus one ormore additional stabilizing agents described herein, in combination withthe buffering agent disclosed herein, i.e., an acid substantially freeof its salt form, the salt form of the acid, or a mixture of the acidand its salt form, leads to an increase in stability of the liquidpolypeptide-containing pharmaceutical composition during storage. Thus,the invention also provides a method for increasing storage stability ofa liquid pharmaceutical composition when that composition comprises apolypeptide that forms aggregates during storage in a liquidformulation. By “increasing storage stability” is intended thepharmaceutical composition exhibits greater retention of the polypeptideor variant thereof in its proper, nonaggregated, biologically activeconformation during storage, and thus less of a decline in therapeuticefficacy, than does a liquid pharmaceutical composition prepared in theabsence of an amino acid base, or an amino acid base plus one or more ofthe additional stabilizing agents described herein, in combination withthe buffering agent disclosed herein.

Storage stability of a polypeptide-containing pharmaceuticalcompositions made in accordance with the methods of the invention can beassessed using standard procedures known in the art. Typically, storagestability of such compositions is assessed using storage stabilityprofiles. These profiles are obtained by monitoring changes in theamount of polypeptide present in its nonaggregated, biologically activemolecular form and its potency over time in response to the variable ofinterest, such as pH concentration, stabilizing agent, concentration ofstabilizing agent, etc., as demonstrated in the Examples below. Thesestability profiles can be generated at several temperaturesrepresentative of possible storage conditions, such as freezingtemperature, refrigerated temperature, room temperature, or elevatedtemperature, such as at 40-50° C. Storage stability is then comparedbetween profiles by determining, for example, half-life of thenonaggregated, biologically active molecular form of the polypeptide ofinterest. By “half-life” is intended the time needed for a 50% decreasein the nonaggregated, biologically active molecular form of thepolypeptide of interest. Compositions comprising arginine base and anacid substantially free of its salt form prepared in accordance with themethods of the present invention will have a half life that is at leastabout two-fold to about ten-fold greater, preferably at least aboutthree-fold to at least about 10-fold greater, more preferably at leastabout four-fold to about ten-fold greater, most preferably at leastabout five-fold to about ten-fold greater than the half-life of a liquidcomposition prepared in the absence of an amino acid base base, or anamino acid base plus one or more of the additional stabilizing agentsdescribed herein, in combination with an acid substantially free of itssalt form, the salt form of the acid, or a mixture of the acid and itssalt form. For purposes of the present invention, a pharmaceuticalcomposition having increased storage stability as a result of beingprepared in accordance with the present invention is considered a“stabilized” pharmaceutical composition. Such a stabilized compositionpreferably has a shelf-life of at least about 18 months, more preferablyat least 20 months, still more preferably at least about 22 months, mostpreferably at least about 24 months when stored at 2-8° C.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

IL-2 is a potent mitogen that stimulates T-cell proliferation. It haswide therapeutic application as a treatment for cancer metastasis, as anadjuvant for cancer therapy, and as a conjunctive agent for infectiousdiseases.

With the progress of various clinical trials using IL-2 therapy, it hasbeen realized that development of a stable liquid formulation for thisprotein is highly desirable. Such a formulation would be more versatilethan traditional lyophilized formulations, as it could be supplied indifferent strengths according to various dosing regimens. A liquidformulation would also be more convenient to administer, as noreconstitution would be needed. Such a formulation may be supplied inprefilled, ready-to-use syringes, or as multidose preparations if foundcompatible with bacteriostatic agents.

It has been reported that IL-2 in liquid formulations degrades via atleast three pathways during storage: aggregation, methionine oxidation,and deamidation (Kunitani et al. (1986) J. Chromatography 359:391-401;Kenney et al. (1986) Lymphokine Res. 5:523-527). In addition, IL-2 issusceptible to acute damage caused by freezing and mechanical shearingstress. Therefore, IL-2 formulation development needs to address bothacute damage and chronic degradation.

Accordingly, a new stable, monomeric rhIL-2 formulation has beendeveloped. In this formulation, the protein molecules are present insolution in their monomer form, not in an aggregated form. Hencecovalent or hydrophobic oligomers or aggregates of rhIL-2 are notpresent. The formulation contains several stabilizing agents, mostimportantly arginine and methionine, to stabilize the protein againstphysical and chemical damages such as aggregation, methionine oxidation,and deamidation during long-term storage. In addition, a nonionicsurfactant, polysorbate 80, has been included in the formulation toprohibit the protein from acute damage caused by freeze-thaw andmechanical shearing stress. As shown in the following examples, additionof the stabilizing agents of the invention to the rhIL-2 formulationincreases its storage stability.

The IL-2 molecule used in these examples is the recombinant human IL-2mutein, aldesleukin, with cysteine-125 replaced by serine (des-alanyl-1,serine-125 human interleukin-2). It is expressed from E. coli, andsubsequently purified by diafiltration and cation exchangechromatography as described in U.S. Pat. No. 4,931,543. Purified bulkfor development use was at about 3 mg/ml of IL-2 and was formulatedeither in 10 mM sodium citrate at pH 6, 200-250 mM NaCl (the CM poolbuffer) or in a buffer containing 10 mM sodium succinate at pH 6 and 150mM L-arginine.

Tissue factor pathway inhibitor (TFPI) is another protein that exhibitsdegradation by aggregation during storage in a liquid pharmaceuticalformulation (Chen et al. (1999) J. Pharm. Sci. 88:881-888). Example 8below is directed to liquid formulations that demonstrate theeffectiveness of using an acid in its free base form to decreaseaggregate formation and a buffering system supplied by an acidsubstantially free of its salt form.

The following protocols were used in the examples to determine effect ofa particular stabilizing agent on IL-2 or TFPI degradation, and hencestability of this protein during storage in liquid formulations.

UV Absorbance Measurement

UV absorbance of protein solutions was measured using a Hewlett PackardDiode Array spectrometer (Model 8452). The instrument was blanked withthe appropriate formulation buffer. Absorbance at 280 nm was recordedusing a 1.0 cm pathlength quartz cuvette. The extinction coefficient of0.70 (mg/ml)⁻¹cm⁻¹ was used to convert the absorbance data to IL-2concentration in mg/ml.

RP-HPLC

RP-HPLC was performed on a Waters 626 LC system equipped with a 717autosampler (Waters Corporation, Milford, Me.) using a Vydac 214BTP54 C₄column and a Vydac 214GCC54 pre-column (Separations Group, Hesparia,Calif.). The columns were initially equilibrated with a mobile phase A(10% acetonitrile, 0.1% TFA). Then 20 μg of an IL-2 sample was loaded,and the protein was eluted by applying a mobile phase B (100%acetonitrile, 0.1% TFA) from 0 to 100% in 50 minutes at a flow rate of1.0 ml/min. The main soluble IL-2 species was eluted at approximately 32min and detected by UV 214 nm using a Waters 486 detector. Dataacquisition and processing were performed on a Perkins-Elmer Turbochromsystem (PE Nelson, Cupertino, Calif.).

This RP-HPLC method detects the main monomeric IL-2 species as peak B, amethionine oxidative species (mainly oxidized Met¹⁰⁴) as peak A, adeamidated species (probably Asn⁸⁸) as peak B′, and other unknownspecies eluting either earlier or later than these peaks.

SEC-HPLC

Size exclusion HPLC was performed on a TOSOHAAS G2000SWx1 column and aTSK SWx1 guard column (TOSOHAAS, Montgomeryville, Pa.). A single mobilephase containing 10 mM sodium phosphate at pH 7 and 200 mM ammoniumsulfate was applied at a flow rate of 1.0 ml/min. The monomer IL-2species was eluted at approximately 14 min and detected by UV 214 nmusing a Waters 486 detector. Data acquisition and processing wereperformed on a Perkin-Elmer Turbochrom system.

Using a native SEC-HPLC protocol specially developed to monitor IL-2,the rhIL-2 eluted mainly as a single species, likely in the monomericform since addition of aggregation dissociation agents, such as SDS,urea, and DTT, did not affect the elution of this species.

IEX-HPLC

Ion exchange (IEX)-HPLC was performed on a Pharmacia Mono-S HR 5/5 glasscolumn using a Waters 626 LC system with a 717 heater/cooler autosampleras described in Chen et al. (1999) J. Pharm. Sci. 88:881-888. The columnwas equilibrated with 80% mobile phase A (70:30 v/v, 20 mM sodiumacetate:acetonitrile at pH 5.4) and 20% mobile phase B (70:30 v/v, 20 mMsodium acetate and 1 M ammonium chloride:acetonitrile at pH 5.4). Afterinjection, recombinant human (rh) TFPI was eluted by increasing mobilephase B to 85% in 21 minutes at a flow rate of 0.7 ml/minute. The rhTFPIeluted at approximately 16.5 minutes as a single peak and was detectedby UV absorbance at 280 nm with a Waters 486 absorbance detector. Dataacquisition and processing were performed on a Perkin-Elmer Turbochromsystem. Protein concentration was estimated by integrating the peak areaand comparing it with a standard curve generated from samples of knownconcentrations.

SDS-PAGE

SDS-PAGE was performed according to the Laemmli protocol. About 5 μg ofIL-2 was loaded into each lane of a pre-cast 18% Norvex Tris-glycine geland electrophoresis was carried out at 100 Volts. Protein bands werestained by the Coomassie blue dye and were analyzed by a MolecularDynamics densitometer equipped with the Imagequan T system (MolecularDynamics, Sunnyvale, Calif.).

HT-2 Cell Proliferation and MTT Stain for IL-2 Bioactivity

The potency of IL-2 was determined by an in vitro bioassay using HT-2cell proliferation and MTT stain (Gillis et al. (1978) J. Immunology120:2027-2032; Watson (1979) J. Exp. Med. 150(6):1510). Briefly, 1×10⁴of murine HT-2 cells, which were IL-2 dependent for growth, were loadedinto a well of tissue culture plate containing standards, controls, orsamples. After 22 to 26 hr incubation at 37° C., MTT stain was addedinto the wells and incubation was continued at 37° C. for 3 to 4 hr.Then 20% SDS was added for destaining overnight at room temperature.Absorbance of the wells was read at 570 nm and converted to the IL-2bioactivity based on the WHO International standards.

pH and Osmolarity Measurements

The solution pH of the various formulations was measured by a pH meterfrom Orion (Model 611, Orion Research Incorporated Laboratory ProductsGroup, Boston, Mass.). The pH meter was calibrated by the two-buffercalibration procedure suggested by the manufacturer using a pH 4standard (Fisher Scientific, Cat. No. SB101-500) and a pH 7 standard(Fisher Scientific, Cat. No. SB107-500).

The solution osmolarity of these formulations was measured by a VaporPressure Osmometer from Wescor Model 5500, Wescor Inc., Logan, Utah).The osmometer was calibrated by two standards supplied by themanufacturer: 290 mmol/kg standard (Wescor, Reorder No. OA-010) and1,000 mmol/kg standard (Wescor, Reorder No. OA-029).

These protocols were used to quantify the effects of various stabilizingagents on rhIL-2 degradation via protein aggregation, methionineoxidation, and deamidation.

Example 1 Effects of Various Solubilizing Agents on Protein Aggregationand Storage Stability of rhIL-2

Protein aggregation is the major degradation pathway for rhIL-2 inliquid media ranging from mildly acidic to alkaline pH conditions.rhIL-2 in solutions formulated with these pH conditions, when stored atelevated temperatures, quickly results in protein aggregation, whichleads to visible precipitation. The visible precipitated protein isremovable by filtration through a 0.2 μm filter. The remaining solubleprotein in solution can be quantified by a number of analytical assayssuch as RP-HPLC, SEC-HPLC, and UV absorbance. Aggregation also resultsin a decrease in bioactivity, which can be determined by the in vitrobioassay described herein.

Using the analytical procedures described herein, the storage stabilityof rhIL-2 under several conditions was followed by monitoring changes inthe amount of soluble rhIL-2 as a function of incubation time atelevated temperatures.

1.A. Effects of Sugars and Amino Acids

The effect of sugars on rhIL-2 storage stability was examined forsorbitol, sucrose, and mannitol in formulations containing 0.2 mg/mlrhIL-2, 10 mM sodium succinate at pH 6, and 270 mM of one of thesesugars. The amount of soluble rhIL-2 remaining in stability samples wasplotted against incubation time as shown in FIG. 1. The curves ofsucrose and mannitol superimposed on each other indicate their effectson IL-2 storage stability are similar. The curve for sorbitol isslightly higher than the other two sugars, suggesting sorbitol has aslightly greater stabilization effect than the other two sugars.

The effect of amino acids on storage stability is shown in FIG. 2.Formulations contained 0.1 mg/ml IL-2, 10 mM sodium succinate at pH 6,and 150 mM of one of the nine amino acids chosen. As shown in FIG. 2,the stability rank is Arg>Asp>Lys>Met>Asn>Leu=Ser=Pro=Gly.

The stabilizing effect of sorbitol and arginine was confirmed in furtherstudies, which showed that rhIL-2 storage stability was affected in aconcentration dependent manner. Thus, rhIL-2 storage stability isenhanced when sorbitol concentration is increased from 50 mM to 150 mM,and finally to 270 mM (FIG. 3). Similarly, rhIL-2 storage stabilityincreases with increasing concentration of arginine in the formulation(FIG. 4).

1.B. Effect of Formulation pH

The pH storage stability profiles of rhIL-2 in formulations containingNaCl, sorbitol, and arginine were examined. Half-lives for the remainingsoluble IL-2 at 50° C. are plotted against pH in FIG. 5. Half-life(t_(1/2)) was defined here as the time needed for a 50% decrease insoluble protein in stability samples. Greater half-life indicatesgreater storage stability.

As shown in FIG. 5, pH optimum for stabilizing rhIL-2 against proteinaggregation depends upon the stabilizing agent present in solution.Maximum storage stability of rhIL-2 in NaCl is reached at pH 4, whererhIL-2 has a half-life at 50° C. of approximately 23 days. rhIL-2 insorbitol formulations shows increased stability as pH decreases, withthe maximum stability occurring at pH 5, where the half-life at 50° C.is about 26 days. The greatest stability (i.e., longest half-life) couldbe achieved with arginine as the stabilizing agent, in a formulationhaving a pH of about 6.0, where the protein's half-life at 50° C. wasabout 32 days. These results suggest arginine is a preferred stabilizingagent relative to sorbitol or NaCl, as optimum pH for proteinstabilization occurs at a more physiologically acceptable pH.

1.C. Effect of Buffer System

It is quite customary to use a 10 mM buffer system in a formulation toprovide a proper pH and to maintain a certain amount of bufferingcapacity. For instance, a formulation pH of 5.8 can be achieved by using10 mM of a mixture of succinic acid and its salt form, such as sodiumsuccinate. If such a buffer system is selected, 150 mM arginine HCl, butnot 150 mM arginine base, can be used as the primary stabilizing agentin the formulation, since 150 mM arginine base would prohibit pH beingadjusted down to 5.8 by the 10 mM buffer.

However, arginine HCl gives rise to a higher osmolarity than doesarginine base. Thus, a formulation containing 150 mM arginine HCladjusted to pH 5.8 with 10 mM succinic acid and sodium succinate bufferis already close to isotonicity, having an osmolarity of about 253mmol/kg, and a half-life at 50° C. of about 8 days (Table 1). Yet ahigher concentration of arginine in the formulation is desirable, asstorage stability increases with increases in this stabilizing agent.When the concentration of arginine is increased to 230 mM with additionof arginine HCl and pH is adjusted to 5.8 with 10 mM succinic acid andsodium succinate buffer, the half-life at 50° C. is doubled (about 17days), yet the solution is hypertonic, having an osmolarity of about 372mmol/kg.

When succinic acid served as the buffering system to adjust solution pHto 5.8 and arginine was present as arginine base, increasingconcentration of arginine base to 230 mM resulted in a similar doublingof the half-life at 50° C., increasing it to about 16 days. However,this increase in storage stability was achieved while keeping thesolution nearly isotonic, with the formulation having an osmolarity ofabout 271 mmol/kg (see Table 1). In this manner, 230 mM arginine basecould be used in the formulation to increase storage stability of rhIL-2without exceeding isotonicity of the formulation. TABLE 1 Solutionosmolarity and storage stability of rhIL-2 formulations. The storagestability is displayed in half-lives (t_(1/2)) for remaining solublerhIL-2 measured by RP-HPLC after storage at 50° C. Arginine HClformulations contained 0.5 mg/ml rhIL-2, 1 mM EDTA, and 150 mM or 230 mML-arginine HCl and 10 mM of succinic acid and sodium succinate to adjustpH to 5.8. Arginine base formulations contained 0.5 mg/ml rhIL-2, 1 mMEDTA, and 150 mM or 230 mM L-arginine base and 81 mM or 128 mM succinicacid to adjust pH to 5.8. Formulation Osmolarity t_(1/2) at 50° C. (allcontained 1 mM EDTA and at pH 5.8) (mmol/kg) (day) 150 mM ArgHCl 10 mMNa Succinate/ 253 8.0 Succinic acid 150 mM ArgBase, 81 mM Succinic acid192 9.9 230 mM ArgHCl 10 mM Na Succinate/ 372 16.9 Succinic acid 230 mMArgBase, 128 mM Succinic acid 271 16.0

These two pH adjustment methods were examined with other buffer systems(Table 2). When 150 mM arginine HCl was used in the formulation and pHwas adjusted to 5.8 by 10 mM of an acid and its sodium salt, allformulations were below isotonic, which is approximately 290 mmol/kg.The half-life at 50° C. for the rhIL-2 in these formulations ranged fromabout 15 to about 20 days. When 230 mM arginine base was used in theformulation and pH was titrated to 5.8 using an acid substantially freeits salt form as the buffer system, formulations having pH adjusted withcitric acid or succinic acid showed solution osmolarities still belowisotonic, while other formulations were either slightly above isotonic,as in the case of phosphoric acid, or hypertonic, as in the case ofglutamic or acetic acid. However, the half-life at 50° C. for allformulations was increased to above 30 days. Thus, by using citric acidor succinic acid, both substantially free of their salt forms, as thebuffering system, the concentration of arginine could be increased to230 mM using arginine base, resulting in increased storage stability ofrhIL-2. TABLE 2 Solution osmolarity and storage stability of rhIL-2formulations. The storage stability is displayed in half-lives (t_(1/2)) for remaining soluble rhIL-2 measured by RP-HPLC after storage at 50°C. All formulations contained 0.2 mgl/ml rhlL-2, 5 mM methionine, 1 mMdisodium EDTA, 0.1% polysorbate 80 and 150 mM L-arginine HCI with pHadjusted to 5.8 by 10 mM of an acid and its sodium salt or 230 mML-arginine base with pH adjusted to 5.8 by titrating with an acidsubstantially free of its salt form. Osmolarity t_(1/2) Formulation(mmol/kg) (day) 150 mM ArgHCI, 10 mM Sodium Citrate/Citric 248 20.5 Acid230 mM ArgBase, 86 mM Citric Acid 228 36.1 150 mM ArgHCI, 10 mM SodiumSuccinate/ 257 19.1 Succinic Acid 230 mM ArgBase, 128 mM Succinic Acid285 29.2 150 mM ArgHCI, 10 mM Sodium Phosphate/ 260 15.6 Phosphoric Acid230 mM ArgBase, 193 mM Phosphoric Acid 329 29.9 150 mM ArgHCI, 10 mMSodium Glutamate/ 264 14.6 Glutamic Acid 230 mM ArgBase, 225 mM GlutamicAcid 407 47.5 150 mM ArgHCI, 10 mM Sodium Acetate/ 259 20.8 Acetic Acid230 mM ArgBase, 250 mM Acetic Acid 408 31.91.D. Effect of Protein Concentration

The effect of protein concentration on storage stability was examined ina formulation containing 10 mM sodium succinate at pH 6 and 150 mMarginine. As shown in FIG. 6, in which the half-life at 50° C. forsoluble rhIL-2 was plotted against initial protein concentration, rhIL-2storage stability increases as protein concentration decreases. Thisfinding is in agreement with the experimental observation thataggregation is the major degradation pathway for rhIL-2 in liquidformulations.

1.E. Effect of the Nonionic Surfactant Polysorbate 80

Effect of polysorbate 80 (TWEEN 80® or TW 80™) on rhIL-2 storagestability was tested in a formulation containing 0.5 mg/ml IL-2, 230 mMarginine base, 128 mM succinic acid to adjust pH to 5.8, 1 mM EDTA and0, 0.02, and 0.1% polysorbate 80. As shown in Table 3, both formulationscontaining polysorbate 80 exhibit a reduction in the half-life ofsoluble IL-2 at 50° C., from 16 days to about 9 days as measured byRP-HPLC. Thus, including polysorbate 80 in the formulation would beperceived as unfavorable based solely on its effect on proteinaggregation. However, this agent has a stabilizing effect against acuteprotein damage associated with freeze-thaw and mechanical shearing thatis beneficial during processing of liquid formulations containing thisprotein, as disclosed in the examples below.

Storage stability of two sorbitol-based formulations was also examined.Although their half-lives by RP-HPLC and bioactivity were comparable tothe arginine formulations, the half-lives estimated from SEC-HPLCwere-much smaller, suggesting a larger portion of the rhIL-2 protein inthese formulations is probably present in soluble aggregated forms.TABLE 3 Half-lives (t_(1/2)) or remaining soluble rhIL-2 (peak B)measured by RP-HPLC, SEC-HPLC, and the in vitro bioassay in rhIL-2formulations stored at 50° C. Formulation T_(1/2) at 50° C. (day) (allat pH 5.8 and 1 mM EDTA) RP SEC Bioactivity 230 mM ArgBase, 128 mM Sucacid, pH 5.8 16.0 21.3 25.6 230 mM ArgBase, 128 mM Suc acid, 9.7 12.4 NA0.02% Tw 80, pH 5.8 230 mM ArgBase, 128 mM Suc acid, 9.1 12.2 24.5 0.1%Tw 80, pH 5.8 270 mM sorbitol, 10 mM NaSuc, 31.7 3.6 NA 0.1%, Tw 80, pH4.5 270 mM sorbitol, 10 mM NaSuc, 14.4 2.4 21.3 0.1% Tw 80, pH 5.0

In Table 3, the half-life for an arginine base-succinic acid rhIL-2formulation as determined by the RP-HPLC method is slightly smaller thanthat determined by the SEC-HPLC method, and is much smaller than thatdetermined by the in vitro bioassay method. The elution of the mainrhIL-2 species on SEC-HPLC was evaluated to further examine thesedifferences. Samples with and without treatment of SDS, urea, and DTTshowed no change in the elution time for the main species, indicatingthat the rhIL-2 in these formulations was present as a monomericspecies. However, the SEC-HPLC protocol might not be able to distinguishother monomeric species, for example, the peak A methionine oxidativespecies, from the major monomeric intact species, the peak B species.Therefore, a small difference in determining the half-life would beexpected.

The in vitro bioassay used to determine bioactivity in the datapresented in Table 3 was carried out with 0.1% SDS in the assay diluent.Thus, prior to applying samples to the tissue culture plate to interactwith murine HT-2 cells, the samples were diluted with assay diluentcontaining 0.1% SDS. It was possible that dilution with SDS might havedissociated some rhIL-2 aggregate in these samples back to the monomericform, resulting in an overestimate of the bioactivity of a givenformulation. Therefore, stability samples were assayed using assaydiluent with (+S) and without (−S) the addition of SDS. Samples werealso assayed with (+F) and without (−F) a 0.2 μm filtration treatment,since filtration is able to remove large protein aggregates as judged byvisual inspection. Bioactivity values measured for samples with thesetreatments are shown in Table 4 along with storage stability resultsobtained using the RP-HPLC protocol for comparison. Values are presentedas a percentage of the bioactivity values obtained in similar samplesstored at −70° C.

In general, the formulations diluted with SDS and not filtered prior tocontact with HT-2 cells show higher bioactivity values than thoseformulations diluted without SDS in the assay diluent and filtered priorto running the assay. Among these bioactivity results, those obtainedusing filtration and diluting with no SDS are quite comparable withRP-HPLC results. Thus, this method is recommended for the truebioactivity measurement for monomeric rhIL-2. TABLE 4 Comparison ofresults between RP-HPLC analysis for soluble rhIL-2 and in vitrobioactivity analysis for stability samples stored 2 wk at 40° C. or 50°C. All results are presented as percentages of those obtained for theirrespective −70° C. samples. Formulations contained 0.5 mg/ml rhIL-2, 230mM L-arginine base, 128 mM succinic acid at pH 5.8, and 1 mM EDTA, with(#1) and without (#2) 0.1% polysorbate 80. Samples for the bioassay weretreated with and without a 0.22 μm filtration before dilution usingdiluents with and without 0.1% SDS. % Bioactivity % RP Sample(percentage to −70° C. samples) (percentage to HPLC −F+S^(a) +F+S^(a)−F−S^(a) +F−S^(ab) −70° C. samples) #1 at 40° C. 106 100 104 100 101 #1at 50° C. 92 79 60 53 58 #2 at 40° C. 101 69 106 112 98 #2 at 50° C. 6038 62 47 42^(a)“−F” for non-filtered, “+F” for filtered, “−S” for diluent withoutSDS and “+S” for diluent with SDS.^(b)This is the recommended protocol for monomeric IL-2.1.F. Preservative Compatibility

Preservative compatibility was investigated in the need to develop amultidose formulation. Effect of preservatives on IL-2 stability wasevaluated in two accelerated studies. Study 1 screened benzyl alcohol,m-cresol, and phenol in a formulation containing 0.2 mg/ml IL-2, 10 mMsodium succinate at pH 6 and 160 mM arginine. Study 2 screenedbenzethonium chloride, benzalkonium chloride, methyl paragen/propylparben, and chlorobutanol in a formulation containing 0.2 mg/ml Il-2,230 mM L-arginine base, 128 mM succinic acid at pH 5.8, 1 mM disodiumEDTA, 0.1% polysorbate 80. The half-lives for soluble rhIL-2 measured byRP-HPLC for these formulations stored at 40° C. are presented in Table5.

All tested preservatives decreased rhIL-2 stability at the elevatedtemperature. In study 1, the half-life for the soluble rhIL-2 was about74 days at 40° C. without any of the preservatives. Addition of benzylalcohol or m-cresol or phenol reduced the half-life significantly by 5-to 10-fold. In study 2, the effects of preservatives were much lessintense. The half-life for the soluble rhIL-2 decreased less than halffor benzethonium chloride and decreased more than 2-fold for otherpreservatives. Overall, benzethonium chloride has the least reduction inrhIL-2 stability among the preservatives tested. TABLE 5 Half-life(t_(1/2)) of the soluble rhIL-2 at 40° C. for formulations containingpreservatives. Study 1: formulations contained 0.2 mg/ml rhIL-2, 10 mMsodium succinate at pH 6, 150 mM L-arginine and preservatives. Study 2:formulations contained 0.2 mg/ml rhIL-2, 230 mM L-arginine base, 128 mMsuccinic acid at pH 5.8, 1 mM disodium EDTA, 0.1% polysorbate 80, andpreservatives. Preservative t_(1/2) at 40° C. (day) Study 1 Nopreservative 74.0  0.9% (w/v) benzyl alcohol 16.0 0.25% (w/v) m-cresol7.5  0.5% (w/v) phenol 11.0 Study 2 No preservative 261.0 0.01% (w/v)benzethonium chloride 185.0 0.01% (w/v) benzalkonium chloride 67.0 0.18%(w/v) methyl paraben and 0.02% 113.0 (w/v) propyl paraben  0.5% (w/v)chlorobutanol 84.0

Although preservatives had a pronounced destabilizing effect on rhIL-2at the elevated temperature, their effects on rhIL-2 short-term storagestability at 4° C. or 25° C. were also examined. A new study was carriedout to examine six preservatives in a same formulation containing 0.2mg/ml rhIL-2, 230 mM L-arginine base, 128 mM succinic acid, 1 mM EDTA, 5mM methionine, and 0.1% polysorbate 80 at pH 5.8. Table 6 reportsresults of the amount of soluble rhIL-2 retained after one year ofstorage. All formulations, when compared to the control, show nosignificant loss in the soluble rhIL-2 level by both the RP-HPLC and thein vitro bioassay, with the exception of the formulation containing0.25% m-cresol, which showed detectable loss. TABLE 6 One year storagestability of preservative-containing formulations at 4° C. or 25° C.presented in percentage of total soluble rhIL-2 remaining as determinedby the RP-HPLC integrated peak areas and the in vitro bioactivity. Thecontrol formulation contained 0.2 mg/ml IL-2, 230 mM L-arginine base,128 mM succinic acid, 1 mM EDTA, 5 mM methionine, and 0.1% polysorbate80 at pH 5.8. % of Initial IL-2 In vitro bioactivity by RP-HPLC (×10°IU/ml) Preservative 4° C. 25° C. t = 0 1 yr/4° C. 25° C./I yr Control102 93 3.8 3.3 2.5  0.9% benzyl alcohol 100 88 4.8 4.0 5.0 0.25%m-cresol 98 60 5.8 2.6 2.5  0.5% phenol 99 87 4.7 3.9 3.2 0.01%benzalkonium 102 93 5.5 3.9 4.4 chloride 0.01% benzethonium 98 89 5.43.2 4.7 chloride  0.5% chlorobutanol 99 91 5.1 3.6 4.5

Example 2 Effects of Various Factors on Methionine Oxidation and StorageStability of rhIL-2

Methionine oxidation in IL-2 has been characterized previously (Kunitaniet al. (1986) J. Chromatography 359:391-402; Sasaoki et al. (1989) Chem.Pharm. Bull. 37(8):2160-2164). Il-2 has four methionine residues atresidue positions 23, 39, 36 and 104 on the polypeptide chain. Amongthese, Met¹⁰⁴ is on the protein surface and is most oxidative. Thismethionine oxidative species can be resolved as an earlier elutingspecies (peak A) to the main IL-2 species (peak B) from the RP-HPLCchromatogram. Met²³ and Met³⁹ are less prone to oxidation, which onlyoccurs under extreme oxidative conditions, probably due to theirexistence in the interior of the protein molecule. Oxidative species ofthese MET residues may elute as earlier species than the Met¹⁰⁴ onRP-HPLC. Met⁴⁶ is buried deep inside of the protein molecule and is noteasily oxidized unless the protein completely unfolds.

Investigation of methionine oxidation in IL-2 concentrated on oxidationof Met¹⁰⁴, as it is the most susceptible methionine residue to oxidationand prevention of its oxidation will also prohibit oxidation of othermethionine residues.

2.A. Effect of pH

Methionine oxidation was studied at a pH range from 3 to 9 informulations containing 0.2 mg/ml IL-2, 150 mM NaCl, and 10 mM ofvarious buffer species. Methionine oxidation in these formulations wasexamined by quantifying percentage of the peak A species (i.e., theMet¹⁰⁴ oxidized species) in IL-2 samples at t=0 and t=3 months. Asreported in Table 7, effect of pH is not noticed at t=0 except in thesample buffered by citrate at pH 6. Compared with other samples, whichhave 5% of the peak A species, the citrate sample showed an increase inthe level of peak A to 6%.

At t=3 months, the level of peak A is increased at higher pH conditions,suggesting a base-catalyzed mechanism for methionine oxidation ofMet¹⁰⁴. At pH 6, succinate is a better buffer than citrate in minimizingmethionine oxidation, as lower values of peak A are observed in thesuccinate formulation. TABLE 7 RP-HPLC analysis of methionine oxidation,expressed as percentage of the total amount of soluble IL-2 (peak A +peak B) present as the methionine-oxidized peak A species (% peak A oftotal soluble IL-2) for pH 3 to pH 9 formulation samples at t = 0 and 3months. Fomulations contained 0.2 mg/ml IL-2, 150 mM NaCl, and pHadjusted by 10 mM of various buffer species. % peak A of total solubleIL-2 t = 3 months Buffer and pH t = 0 −70° C. 4° C. 25° C. 40° C. 10 mMGlycine, pH 3 5 4 5 6 7 10 mM Acetate, pH 4 5 5 6 7 6 10 mM Acetate, pH5 NA 7 8 9 9 10 mM Citrate, pH 6 6 6 8 12 14 10 mM Succinate, pH 6 5 6 74 9 10 mM Phosphate, pH 7 5 7 8 11 5 10 mM Borate, pH 9 5 11 15 14 NA2.B. Effects of EDTA, Polysorbate 20, Polysorbate 80, and MgCl₂

The effects of a metal chelator, two nonionic surfactants, and adivalent metal ion on methionine oxidation are reported in Table 8. Thepresence of polysorbate 20 or polysorbate 80 in the formulationsincreases the level of the methionine oxidative species at both t=0 andt=1 month. In contrast, EDTA and MgCl₂ reduce the level of methionineoxidative species after 1 month storage at 40 and 50° C. TABLE 8Percentage of the total amount of soluble IL-2 (peak A + peak B) presentas the methionine-oxidized peak A species (% peak A) in IL-2 samples att = 0 and t = 1 month. The control sample contained 0.2 mg/ml IL-2, 10mM sodium succinate at pH 6, and 150 mM arginine. % peak A (methionineoxidation) t = 1 month Formulation t = 0 −70° C. 4° C. 40° C. 50° C.Control 2.8 3.5 5.1 19.7 36.3 1 mM EDTA 2.5 3.5 5.0 9.5 14.0 0.1%polysorbate 80 5.8 4.3 6.9 21.5 41.4 1 mM EDTA + 0.1% 5.9 4.2 6.9 12.119.2 polysorbate 80 0.1% polysorbate 20 4.1 5.4 9.6 25.3 42.8 5 mM MgCl₂3.9 4.8 6.9 9.7 12.12.C. Effect of Methionine

The addition of methionine in formulations to prevent IL-2 frommethionine oxidation was investigated. Table 9 reports change in peak Aand total amount of soluble IL-2 (peak A+peak B) for formulationscontaining varying amounts of methionine after 2 weeks of storage at 50°C. Increasing of methionine concentration in the formulations reducesthe level of peak A significantly at both t=0 and 2 weeks, while theamount of soluble IL-2 retained after 2 weeks storage is not affected.At 5 mM methionine, a 3-fold decline in peak A is observed at t=2 weeks.Thus, addition of methionine results in a significant reduction inmethionine oxidation of the protein and has little effect on IL-2aggregation. TABLE 9 Change in peak A and in total soluble protein inIl-2 samples at t = 0 and t = 2 weeks at 50° C. Formulations contained0.2 mg/ml IL-2, 230 mM arginine, 128 mM succinic acid at pH 5.8, 1 mMEDTA, 0.1% polysorbate 80 and 0 to 10 mM methionine. % peak A(methionine oxidation) % IL-2 remaining Formulation t = 0 t = 2 wk at50° C. t = 2 wk at 50° C. No Methionine 2.1 6.3 76  1 mM Methionine 1.42.7 77  5 mM Methionine 1.2 2.1 77 10 mM Methionine 1.2 1.9 76

In addition to the high temperature results, level of methionineoxidation at 4° C. and 25° C. after 3 months storage for formulationswith and without 5 mM methionine was also recorded. As shown in Table10, the presence of 5 mM methionine in the formulation results in a3-fold decrease in the level of peak A both at 4° C. and at 25° C. Thus,addition of 5 mM methionine effectively prevented the Met¹⁰⁴ fromoxidation. TABLE 10 Level of methionine oxidation, expressed aspercentage of the total amount of soluble IL-2 (peak A + peak B) presentas the methionine-oxidized peak A species (% peak A of total solubleIL-2) and percentage of soluble IL-2 remaining in samples stored for 3months at either 4° C. or at 25° C. Formulations contained 0.2 mg/mlIL-2, 230 mM arginine, 128 mM succinic acid at pH 5.8, 1 mM EDTA, 0.1%polysorbate 80, and 0 or 5 mM methionine. % peak % IL-2 A of totalremaining soluble IL-2 (main peak) Formulation 4° C. 25° C. 4° C. 25° C.0 mM 2.7 4.1 101 97 5 mM 0.8 1.4 101 1002.D. Effect of Oxygen Removal by Nitrogen Purging and Degassing

Removing oxygen in IL-2 sample vials to minimize methionine oxidationwas tested. Air in the headspace in a 3 cc vial with 1 ml IL-2 samplefill was purged with nitrogen. Dissolved molecular oxygen was removed byvacuum degassing. Table 11 shows the change in peak A and total amountof soluble IL-2 (peak A+peak B) after 1 week of storage at 50° C. forthese samples. Nitrogen purging alone slightly decreases the percentageof the methionine oxidative species from 6.7% to 6.2%. The combinationof solution degassing and nitrogen purging of the headspace furtherreduces the level of peak A by about one percentage point. On the otherhand, the percentage of total amount of soluble IL-2 remains unchangedwith either nitrogen purge or degassing. TABLE 11 Change in percentageof the total amount of soluble IL-2 (peak A + peak B) present as themethionine-oxidized peak A species (% peak A) and in percentage of thetotal amount of soluble IL = 2 remaining in samples withdrawn at t = 0and t = 1 week at 50° C. Formulations contained 0.3 mg/ml of IL-2, 230mM arginine, 128 mM succinic acid, 1 mM EDTA, and 0.1% polysorbate 80. %peak A (methionine oxidation) % IL-2 remaining Formulation t = 0 t = 1wk at 50° C. t = 1 wk at 50° C. Control 3.1 6.7 81 Nitrogen purging 3.16.2 82 Degassing/nitrogen purging 3.0 5.8 812.E. Effect of Preservatives

The effect of preservatives on methionine oxidation was examined. Table12 shows change in peak A for formulation samples with and without oneof the six preservatives after 6 and 12 months storage at 4° C. and at25° C. All formulations containing preservatives showed similar level ofpeak A to the control indicating the preservatives have no detectableeffect on methionine oxidation except in the formulation containing0.25% m-cresol, which showed a significant increase in the peak A level.TABLE 12 Percentage of the total amount of soluble IL-2 (peak A + peakB) present as the methionine-oxidized peak A species (% peak A) invarious preservative-containing formulations stored 6 months and 12months at 4° C. and 25° C. The control formulation contained 0.2 mg/mlIL-2, 230 mM L-arginine base, 128 mM succinic acid, 1 mM EDTA, 5 mMmethionine, 0.1% polysorbate 80, at a pH of 5.8. % peak A (methionineoxidation) 6 months 12 months Preservative t = 0 4° C. 25° C. 4° C. 25°C. Control 1.5 1.6 1.9 1.8 2.6  0.9% benzyl alcohol 1.6 1.7 2.3 1.9 3.30.25% m-cresol 1.6 1.8 6.7 2.1 3.5  0.5% phenol 1.6 1.7 2.4 1.8 3.6 0.01benzalkonium chloride 1.5 1.6 2.0 1.0 3.0 0.01% benzethonium chloride1.5 1.7 2.0 1.8 2.8  0.5% chlorobutanol 1.5 1.6 2.0 1.8 3.2

Example 3 Effect of Various Factors on Deamidation of IL-2

Deamidation of IL-2 has been reported previously (Kunitani et al. (1986)J. Chromatography 359:391-402). Asp⁸⁸ has been discovered to be theprimary site for deamidation in IL-2 (Sasaoki et al. (1992) Chem. Pharm.Bull. 40(4):976-980). Deamidated species can be detected by RP-HPLC as aback shoulder peak (Peak B′) to the main species (peak B). IL-2deamidation was studied in formulations containing arginine, NaCl, andsorbitol. Table 13 shows that deamidated species can be detected only informulations containing sorbitol and NaCl, but not in formulationscontaining arginine, after incubation at elevated temperatures for 2weeks. Therefore, arginine stabilizes IL-2 against degradation viadeamidation. TABLE 13 Deamidation detected by RP-HPLC of peak B′ speciesin formulations containing 0.2 mg/ml IL-2, 10 mM sodium succinate at pH6, and 150 mM arginine, 150 mM NaCl, or 270 mM sorbitol. % Peak B′(Deamidation) at t = 0 and 2 weeks Formulation t = 0 −70° C. 40° C. 50°C. 10 mM NaSuc, 150 mM Arg, pH6 0 0 0 0 10 mM NaSuc, 150 mM NaCl, pH6 00 0 3 10 mM NaSuc, 270 mM Sorbitol, 0 0 2 2 pH6

Example 4 Effect of Freeze-Thawing on IL-2 Stability

Freezing-induced protein damage is usually caused by three mechanisms:(1) the protein is conformationally unstable at cold temperatures (colddenaturation); (2) the protein is susceptible to denaturation onice-water interface; (3) the protein is damaged by changes in saltconcentration or pH shift upon freezing.

In the case of IL-2, protein loss during freeze-thaws is probably causedby denaturation and aggregation on the ice-water interface since thenonionic surfactant polysorbate 80 effectively protected Il-2 fromfreeze-thaw damage. As shown in FIG. 7, the amount of soluble IL-2decreases upon each cycle of freeze-thaw in a formulation containing 0.2mg/ml IL-2, 10 mM sodium succinate at pH 6, and 150 mM arginine. Theaddition of polysorbate 80 in the formulation increases IL-2 stabilityagainst multiple freeze-thaws. When the concentration of polysorbate 80reaches 0.05% and above, IL-2 is fully protected from freeze-thawdamage.

Example 5 Effect of Mechanical Shearing on IL-2 Stability

5.1. Effect of Polysorbate 80, EDTA, Protein Concentration, and FillVolume.

Studies were carried out to examine shear-stress-induced loss of solubleIL-2. Two types of shear stresses were assessed: shaking on an Orbitalshaker (VWR Scientific, Cat. No. 57018-754) and vortexing on a vortexer(Fisher Scientific, model Genie 2, with the speed set at 4). VariousIL-2 formulations were filled 1 ml in 3-cc vials. These vials werestored in a refrigerator (control samples), placed on a laboratory benchovernight (static samples), shaken at 200 RPM overnight (shakingsamples), or vortexed one min (vortexing samples). Table 14 showsresults of change in the amount of soluble IL-2 for these samples.

Compared with the refrigerated control samples, both static samples andshaking samples show no loss in IL-2. Thus, IL-2 is stable at ambienttemperature and is stable to the shaking treatment. On the other hand,when these formulations were subjected to one min vortexing, variousamounts of losses were detected. Formulations containing 0.1 to 0.5mg/ml IL-2 or 1 and 5 mM EDTA or low concentrations of polysorbate 80(0.005 to 0.05%) all show 25-50% loss of soluble IL-2. Therefore, onemin vortexing was more detrimental than overnight shaking to IL-2molecules. The loss of IL-2 could be prevented by increasing theconcentration of polysorbate 80 in the formulation to equal to andgreater than 0.1%. In addition, fully filled vials with no air left inthe headspace also show minimal loss of soluble IL-2 upon one minvortexing, indicating that air-liquid interface was the major factorcausing the damage. TABLE 14 Change in the amount of soluble IL-2 forsamples stored at ambient temperature overnight (Static), shaken at 200RPM overnight (Shaking) and vortexed for 1 min (Vortexing) as comparedwith those stored at 4° C. The control formulation contained 0.2 mg/mlIL-2, 10 mM sodium succinate at pH 6, and 150 mM arginine. Theformulation was filled 1 ml in 3 cc glass vials except for thecompletely filled sample, which had the control sample filled completelyto the top of the 3 cc vials, leaving no air in the headspace. SolubleIL-2 was quantified by RP-HPLC. % Remaining of soluble IL-2 SampleStatic Shaking Vortexing (1 ml fill in 3 cc vials) Overnight Overnight 1min 0.2 mg/ml IL-2 (control) 99.6 100.8 74.7 0.1 mg/ml IL-2 100.3 102.772.0 0.5 mg/ml IL-2 99.7 101.0 68.6 1 mM EDTA 97.6 101.2 59.6 5 mM EDTA99.3 102.7 72.2 0.005% polysorbate 80 100.6 100.0 46.5  0.01%polysorbate 80 99.7 100.4 66.1  0.05% polysorbate 80 99.3 100.3 93.9 0.1% polysorbate 80 99.2 100.0 89.0  0.2% polysorbate 80 99.3 99.2 99.8 0.5% polysorbate 80 99.1 98.4 99.7 1 mM EDTA, 0.1% polysorbate 80 99.6100.1 99.8 Complete filled vials 99.4 100.1 96.55.2 Effect of Arginine

The effect of arginine on IL-2 stability against vortexing damage isreported in Table 15. Increasing arginine concentration from 150 mM to230 mM results in a 3% increase in the amount of soluble IL-2 from 65%to 69% after subjecting to one min vortexing. Thus, arginine has a minoreffect on IL-2 against shear damage although it showed previously agreat stabilization effect on IL-2 against degradation due to aggregateformation.

Effect of polysorbate 80 in the 230 mM arginine formulation was tested.Addition of a low concentration of polysorbate 80 (0.02%) destabilizesIL-2 and addition of a high concentration of polysorbate 80 (0.1%)stabilizes IL-2 against vortexing damage. TABLE 15 Percent remaining ofsoluble IL-2 in various formulations upon 1 min vortexing as analyzed byRP-HPLC Formulation % IL-2 (all contains 0.5 mg/ml IL-2 except otherwisenoted) remaining 150 mM Arginine, 10 mM NaSuc, 1 mM EDTA, pH 5.8 65.5150 mM Arginine, 81 mM Suc acid, 1 mM EDTA, pH 5.8 65.3 230 mM Arginine,10 mM NaSuc, 1 mM EDTA, pH 5.8 69.0 230 mM Arginine, 128 mM Suc acid, 1mM EDTA, pH 5.8 68.9 230 mM Arginine, 128 mM Suc acid, 1 mM EDTA, 55.10.02% Tw 80, pH 5.8 230 mM Arginine, 128 mM Suc acid, 1 mM EDTA, 99.50.1% Tw 80, pH 5.85.3. Shipping Study

Shear damage on IL-2 during product shipment was investigated in a realshipping study. IL-2 was prepared in an arginine formulation and a NaClformulation, both with varying amounts of polysorbate 80. These IL-2samples were shipped on ice by air from Emeryville, Calif., to St.Louis, Mo., and from St. Louis back to Emeryville. FIG. 8 shows RP-HPLCanalysis of the amount of soluble IL-2 in these samples. Without thepresence of polysorbate 80 in the formulation, about 10% loss of IL-2 isobserved in both arginine and NaCl formulated samples. The stabilitydifferences between the arginine formulation and the NaCl formulationare negligible, around 1%. With the presence of polysorbate 80 in theformulation, loss of IL-2 is reduced. At 0.1% polysorbate 80, no loss isobserved, indicating that IL-2 was fully protected at this surfactantconcentration. Thus, 0.1% polysorbate 80 is effective in the formulationto prevent IL-2 from acute shear damage during shipping.

In conclusion, arginine may serve as a primary stabilizing agent inliquid IL-2 pharmaceutical formulations during long-term storage todecrease IL-2 aggregation and deamidation. To further increase arginineconcentration in the formulation, and thus to achieve a greater IL-2stability but still maintain the solution isotonicity, succinic acid ispreferably used to titrate arginine base to pH 5.8. In addition,methionine and EDTA may be included in the formulation to preventmethionine oxidation of the protein. Finally, a nonionic surfactant,such as polysorbate 80, may be included in the formulation to preventIL-2 from damage by freeze-thawing and mechanical shearing.

Example 6 Preservative Effectiveness Test

Several formulations containing antimicrobial preservatives have beensubjected to a United States Pharmacopoeia (USP) preservative efficacytest. The results are presented in Table 16. The control sample withoutpreservative failed the test while all formulations containing apreservative passed the test. TABLE 16 USP preservative efficacy testfor rhIL-2 formulations. The control formulation contained 0.1 mg/m1rhIL-2, 230 mM L-arginine base, 128 mM succinic acid, 1 mM EDTA, 5 mMmethionine, 0.1% polysorbate 80, at a pH of 5.8. PRESERVATIVE USP TESTControl fail 0.9% benzyl alcohol pass 1.3% benzyl alcohol pass 1.7%benzyl alcohol pass 0.5% chlorobutanol pass 0.5% phenol pass

Example 7 Pain Producing Properties

A rat model developed at University of Florida, College of Dentistry,OMSDS Division of Neuroscience, was used to assess pain-producingproperties, more particularly the burning and stinging pain produced byformulations. The model is based on an assay of the current induced insensory cells that carry pain messages. To conduct the assay, sensorycells (rat dorsal root ganglion) are isolated in a recording chamber.Recordings are made from individual cells that are pre-selected basedupon nociceptive (pain-inducing) criteria. Burning pain, stinging pain,and standardized pain scores are computer for the tested formulation. Aburning pain score is defined by the response to the test formulationrelative to capsaicin (500 nM). Capsaicin is well known for its capacityto produce intense burning pain in humans (Cooper et al. (1996) Pain24:93-116). A stinging pain score is computed as the ratio of thecurrent produced by the test formulation to that produced by a solutionbuffered to pH 5.0. The standardized pain score rates the formulationrelative to normal saline (0.9% NaCl, non-buffered), a common hospitalpharmacy parenteral that is known to produce a stinging sensation.

A liquid L-arginine base-succinic acid formulation has been subjected tothe pain analysis via this model. Results of these two test solutionsare shown in Table 17. Based upon scores calculated for burning pain,stinging pain, and the standardized pain score, the L-arginine-succinicacid formulation exhibited excellent properties in comparison withnormal saline. It was also observed that the test current diminishedduring the application of the formulation (time-dependent decrease)while it did not diminish with normal saline. This demonstrates thatthis formulation is better tolerated than saline as evaluated by thisassay. TABLE 17 Burning, stinging, and standardized pain scores for theliquid formulation and 0.9% NaC1. Standardized Formulation Burning PainStinging Pain pain score 0.9% NaC1 0.084 ± 0.006 2.44 ± 0.62 1.73 ± 0.73RrhIL-2 liquid 0.044 ± 0.019 0.65 ± 0.23 0.16 ± 0.05 formulationThe liquid formulation contained 230 mM L-arginine base, 128 mM succinicacid, 1 mM EDTA, 5 mM methionine, 0.1% polysorbate 80, at a pH of 5.8.

Example 8 Stability Studies with TFPI

Stability and solubility studies of TFPI in various formulations havedemonstrated that L-arginine is a stabilizer (data not shown) to TFPIand charged buffer species such as citrate ions have a more profoundsolubilizing effect. In this study, the effects of L-arginineconcentration and buffering system on TFPI stability in variousformulations were examined. In particular, the influence of bufferingsystem in the form of an acid substantially free of its salt farm versusa mixture of an acid and its salt form were tested as previously notedfor IL-2 formulations in the foregoing examples.

Materials and Methods

A TFPI solution was formulated to 0.6 mg/ml in 20 mM sodium citrate and300 mM L-arginine at pH 5.5. This solution was buffer exchanged viadialysis at 4° C. using the Spectral Por #7 membranes (MWCO 3,500, ID#132-110) to various L-arginine formulations buffered to pH 6.5 by eithercitrate or succinate buffering system. Following dialysis, the TFPIconcentration of each solution was measured using UV/Vis spectroscopy.Each solution was then diluted down to 0.15 mg/ml using the appropriatebuffer. The prepared solutions were then aliquoted (1 ml each) to 3-ccvials for stability storage. Enough vials were set aside at this pointfor the T=0 time point. The rest of the vials were placed in a 50° C.incubator for an accelerated stability study. Time points were thentaken at 3, 7, 14, and 30 days. For analysis at each time point, thecontents of each vial were transferred to a 1.7 ml microcentrifuge tubeand then centrifuged at 10K rpm for approximately 2 minutes. Thecentrifuged supernatant of the samples was taken from this tube foranalysis using IEX-HPLC, which was known from previous studies to be astability indicating assay.

Results and Discussion

TFPI was formulated to 0.15 mg/ml final concentration in variousformulations containing either L-arginine base or L-arginine HCl.L-arginine HCl formulations were buffered to pH 5.5 by 10 mM citric acidor succinic acid in combination with its respective conjugate sodiumsalt. L-arginine base formulations were titrated to pH 5.5 by eithercitric acid or succinic acid. A total of eight studies were carried outas listed below:

-   -   1) 20-150 mM L-arginine HCl buffered to pH 5.5 by 10 mM citric        acid and sodium citrate;    -   2). 20-150 mM L-arginine base titrated to pH 5.5 by citric acid;    -   3) 100-300 mM L-arginine HCl buffered to pH 5.5 by 10 mM citric        acid and sodium citrate;    -   4) 100-300 mM L-arginine base titrated to pH 5.5 by citric acid;    -   5) 20-150 mM L-arginine HCl buffered to pH 5.5 by 10 mM succinic        acid and sodium succinate;    -   6) 20-150 mM L-arginine base titrated to pH 5.5 by succinic        acid;    -   7) 100-300 mM L-arginine HCl buffered to pH 5.5 by 10 mM        succinic acid and sodium succinate; and    -   8) 100-300 mM L-arginine base titrated to pH 5.5 by succinic        acid.

The major degradation pathway for TFPI was previously determined to beprotein aggregation/precipitation (Chen et al. (1999) J. Pharm. Sci.88:881-888). TFPI degradation can be followed by monitoring theremaining soluble protein in stability samples. TFPI solutionsformulated at different L-arginine concentrations were stored at 50° C.for an accelerated stability study. Samples were taken at predeterminedtime intervals. Soluble protein in the samples was separated fromaggregated/precipitated protein through centrifugation in amicrocentrifuge tube. The amount of soluble protein was determined bythe IEX-HPLC method (Chen et al. (1999) J. Pharm. Sci. 88:881-888). Thedata were then fitted as a function of storage time by a singleexponential kinetic equation (Y=YoEXP(−k1t) to calculate the half-lifefor the remaining soluble protein using the KaleidaGraph graphicsoftware (Synergy Software, Reading Pa.).

The half-life (t_(1/2)) values for the remaining soluble TFPI for theformulations buffered by citrate addition of or sodium citrate are shownin Table 18. Those for the formulations buffered by succinic acid orsodium succinate are shown in Table 19. These data demonstrate that thehalf-life value increases with increasing L-arginine concentration inthese formulations. These data are also plotted in FIGS. 9 and 10 forcitrate and succinate buffer systems, respectively. The half-life valueplots as a parabolic curve and increases as a function of arginineconcentration. This establishes that L-arginine is a stabilizer forTFPI.

Between the two buffering systems, the difference in TFPI stabilityappears negligible. Although the citrate buffering system showed morevariability FIG. 9), the two half-life vs. arginine concentration curvesfor the succinate buffering system were essentially superimposable (FIG.10). TFPI achieved similar stability at similar L-arginine concentrationregardless of which of the buffering systems was used for pH adjustment.FIG. 11 also compares the half-life vs. arginine concentration curvesbetween the succinic acid buffer system and the citric acid system. Thisfigure shows that there is no major difference in TFPI stability as longas the arginine concentration remains the same in the formulation. Thesedata demonstrate that the stabilizing effect was mainly contributed fromthe arginine.

However, acid titration with either succinic or citric acid, allows fora greater concentration of arginine in the formulation (and henceincreased stability) while maintaining isotonicity. Thus, for example,both formulations 3-3 and 4-3 in Table 18 have 300 mM L-arginine in theformulations and their half-life values are similar. However, the 3-3formulation used 10 mM citric acid and sodium citrate to buffer 300 mML-arginine HCl to pH 5.5 and had a solution osmolarity of 497 mOsm/kg.This is a hypertonic formulation and is not preferred as an injectableformulation. On the other hand, the 4-3 formulation used 121 mM citricacid in combination with 300 mM L-arginine base to adjust pH to 5.5 andhad a solution osmolarity of 295 mOsm/kg. This formulation is very closeto an isotonic solution (290 mmol/kg), and thus is a more preferredinjectable formulation. If a conventional way for pH adjustment wereused, for instance, with 10 mM citric acid and sodium citrate, one couldonly add slightly more than 150 mM L-arginine to the formulation withoutexceeding isotonicity. The half-life of the 150 mM L-arginineformulation (Code 1-6) is 16 days in comparison with 23 days for the 300mM L-arginine formulation (Code 4-3). Therefore, formulating TFPI withan acid base (i.e., arginine-base) as a stabilizer and a buffercomprising an acid substantially free of its salt form (i.e., succinicacid) provides an effective means to add more stabilizer (i.e.,arginine) to maximize stabilizing effect on TFPI.

CONCLUSION

This example demonstrates that L-arginine stabilizes TFPI by extendingits storage shelf-life. By using acid titration, one can add morearginine to the formulation to maximize the stabilizing effect withoutexceeding isotonicity, which is preferred for injectable formulations.TABLE 18 Stability data for TFPI arginine-citrate pH 5.5 formulations.Osmolarity Code Formulation (mmol/kg) t_(1/2) (Day) 1-1  20 mM L-ArgHCl, 10 mM Citric acid/NaCitrate 66 9.4 1-2  40 mM L-Arg HCl, 10 mMCitric acid/NaCitrate 81 12.6 1-3  60 mM L-Arg HCl, 10 mM Citricacid/NaCitrate 91 10.7 1-4  80 mM L-Arg HCl, 10 mM Citric acid/NaCitrate106 10.9 1-5 100 mM L-Arg HCl, 10 mM Citric acid/NaCitrate 190 12.5 1-6150 mM L-Arg HCl, 10 mM Citric acid/NaCitrate 276 16.0 2-1  20 mM L-ArgBase titrated by 8.9 mM Citric acid 67 5.7 2-2  40 mM L-Arg Basetitrated by 17.8 mM Citric acid 84 15.0 2-3  60 mM L-Arg Base titratedby 26.6 mM Citric acid 95 17.0 2-4  80 mM L-Arg Base titrated by 34.2 mMCitric acid 109 14.6 2-5 100 mM L-Arg Base titrated by 42.6 mM Citricacid 119 18.2 2-6 150 mM L-Arg Base titrated by 62.4 mM Citric acid 14720.4 3-1 100 mM L-Arg HCl, 10 mM Citric acid/NaCitrate 239 14.8 3-2 200mm L-Arg HCl, 10 mM Citric acid/NaCitrate 358 19.6 3-3 300 mM L-Arg HCl,10 mM Citric acid/NaCitrate 497 21.7 4-1 100 mM L-Arg Base titrated by42.2 mM Citric acid 155 16.7 4-2 200 mM L-Arg Base titrated by 81.8 mMCitric acid 224 22.5 4-3 300 mM L-Arg Base titrated by 121 mM Citricacid 295 23.3The half-life (t_(1/2)) was obtained by fitting 50° C. stability datausing a single exponential kinetic equation.

TABLE 19 Stability data for TFPI arginine-succinate pH 5.5 formulations.Osmolarity Code Formulation (mmol/kg) t_(1/2) (Day) 1-1  20 mM L-argHCl, 10 mM Succinic acid/NaSuccinate 66 9.9 1-2  40 mM L-arg HCl, 10 mMSuccinic acid/NaSuccinate 97 11.5 1-3  60 mM L-arg HCl, 10 mM Succinicacid/NaSuccinate 129 15.3 1-4  80 mM L-arg HCl, 10 mM Succinicacid/NaSuccinate 163 16.7 1-5 100 mM L-arg HCl, 10 mM Succinicacid/NaSuccinate 197 20.5 1-6 150 mM L-arg HCl, 10 mM Succinicacid/NaSuccinate 282 21.9 2-1  20 mM L-arg Base titrated by 12.5 mMSuccinic acid 40 6.7 2-2  40 mM L-arg Base titrated by 25.2 mM Succinicacid 62 12.6 2-3  60 mM L-arg Base titrated by 37.5 mM Succinic acid 8516.4 2-4  80 mM L-arg Base titrated by 49.9 mM Succinic acid 107 19.62-5 100 mM L-arg Base titrated by 62.4 mM Succinic acid 129 20.9 2-6 150mM L-arg Base titrated by 91.4 mM Succinic acid 192 23.1 3-1 100 mML-arg HCl, 10 mM Succinic acid/NaSuccinate 207 16.5 3-2 200 mM L-argHCl, 10 mM Succinic acid/NaSuccinate 353 21.6 3-3 300 mM L-arg HCl, 10mM Succinic acid/NaSuccinate 515 21.7 4-1 100 mM L-arg Base titrated by61.3 mM Succinic acid 127 17.0 4-2 200 mM L-arg Base titrated by 122 mMSuccinic acid 256 21.4 4-3 300 mM L-arg Base titrated by 180 mM Succinicacid 363 22.5The half-life (t_(1/2)) was obtained by fitting 50° C. stability datausing a single exponential kinetic equation.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A stabilized liquid pharmaceutical composition comprising: tissuefactor pathway inhibitor (TFPI) or a TFPI variant; arginine base in anamount sufficient to decrease aggregate formation of said TFPI or saidTFPI variant during storage of said composition; methionine in an amountsuch that the ratio of methionine to methionine residues in said TFPI orsaid TFPI variant is from about 1:1 to about 1000:1; and a bufferingagent that is an acid substantially free of its salt form, an acid inits salt form, or a mixture of an acid and its salt form. wherein saidTFPI variant has at least 70% sequence identity with said TFPI.
 2. Thecomposition of claim 1, wherein said TFPI variant has at least 90%sequence identity with said TFPI.
 3. The composition of claim 2, whereinsaid TFPI variant has at least 95% sequence identity with said TFPI. 4.The composition of claim 3, wherein said TFPI variant has at least 98%sequence identity with said TFPI.
 5. The composition of claim 4, whereinsaid TFPI variant is an analogue comprising a native polypeptidesequence and structure of TFPI and having one amino acid insertion. 6.The composition of claim 5, wherein said TFPI variant is a polypeptideconsisting of mature TFPI immediately preceded by an N-terminal alanineresidue.
 7. The composition of claim 1, wherein said TFPI or said TFPIvariant has a concentration in said composition from about 0.01 mg/ml toabout 5.0 mg/ml.
 8. The composition of claim 7, wherein said TFPI orsaid TFPI variant has a concentration in said composition from about0.05 mg/ml to about 2.0 mg/ml.
 9. The composition of claim 8, whereinsaid TFPI or said TFPI variant has a concentration in said compositionfrom about 0.10 mg/ml to about 1.0 mg/ml.
 10. The composition of claim9, wherein said TFPI or said TPFI variant has a concentration in saidcomposition from about 0.10 mg/ml to about 0.60 mg/ml.
 11. Thecomposition of claim 1, wherein said arginine base has a concentrationin said composition from about 100 mM to about 400 mM.
 12. Thecomposition of claim 11, wherein said arginine base has a concentrationin said composition from about 175 mM to about 400 mM.
 13. Thecomposition of claim 12, wherein said arginine base has a concentrationin said composition from about 130 mM to about 375 mM.
 14. Thecomposition of claim 13, wherein said arginine base has a concentrationin said composition from about 150 mM to about 350 mM.
 15. Thecomposition of claim 14, wherein said arginine base has a concentrationin said composition from about 250 mM to about 350 mM.
 16. Thecomposition of claim 14, wherein said arginine base has a concentrationin said composition from about 175 to about 325 mM.
 17. The compositionof claim 16, wherein said arginine has a concentration in saidcomposition from about 180 mM to about 300 mM.
 18. The composition ofclaim 17, wherein said arginine has a concentration of about 300 mM. 19.The composition of claim 1, wherein said composition has a half-life at40-50° C. that is from about two-fold to about ten-fold greater than thehalf-life of said liquid composition, prepared in the absence of saidarginine.
 20. The composition of claim 19, wherein said composition hasa half-life at 40-50° C. that is from about three-fold to about ten-foldgreater than the half-life of said liquid composition, prepared in theabsence of said arginine.
 21. The composition of claim 20, wherein saidcomposition has a half-life at 40-50° C. that is from about four-fold toabout ten-fold greater than the half-life of said liquid composition,prepared in the absence of said arginine.
 22. The composition of claim21, wherein said composition has a half-life at 40-50° C. that is fromabout five-fold to about ten-fold greater than the half-life of saidliquid composition, prepared in the absence of said arginine.
 23. Thecomposition of claim 1, wherein methionine is present in saidcomposition in an amount such that the ratio of methionine to methionineresidues in said TFPI or variant thereof is from about 10:1 to about100:1.
 24. The composition of claim 1, wherein said buffering agentcomprises sodium citrate.
 25. The composition of claim 24, wherein saidbuffering agent has a concentration in said composition of about 20 mM.26. The composition of claim 1, wherein said composition has a pH fromabout 4.0 to about 9.0.
 27. The composition of claim 26, wherein saidcomposition has a pH from about 5.0 to about 6.5.
 28. The composition ofclaim 27, wherein said composition has a pH of about 5.5.